Bio-synthetic matrix and uses thereof

ABSTRACT

A bio-synthetic matrix comprising a hydrogel which is formed by cross-linking a synthetic polymer and a bio-polymer is provided. The matrix is robust, biocompatible and non-cytotoxic and is capable of supporting cell in-growth in vivo. The matrix can be tailored to further comprise one or more bioactive agents. The matrix may also comprise cells encapsulated and dispersed therein, which are capable of proliferating upon deposition of the matrix in vivo. Methods of preparing the bio-synthetic matrix and the use of the matrix in vivo for tissue engineering or drug delivery applications are also provided.

FIELD OF THE INVENTION

The present invention pertains to the field of tissue engineering and inparticular to a bio-synthetic matrix comprising a hydrogel suitable forin vivo use.

BACKGROUND

Tissue engineering is a rapidly growing field encompassing a number oftechnologies aimed at replacing or restoring tissue and organ function.The key objective in tissue engineering is the regeneration of adefective tissue through the use of materials that can integrate intothe existing tissue so as to restore normal tissue function. Tissueengineering, therefore, demands materials that can support cellover-growth, in-growth or encapsulation and, in many cases, nerveregeneration.

Polymer compositions are finding widespread application in tissueengineering. Natural bio-polymers such as collagens, fibrin, alginatesand agarose are known to be non-cytotoxic and to support over-growth,in-growth and encapsulation of living cells. Matrices derived fromnatural polymers, however, are generally insufficiently robust fortransplantation. In contrast, matrices prepared from synthetic polymerscan be formulated to exhibit predetermined physical characteristics suchas gel strength, as well as biological characteristics such asdegradability. Reports that synthetic analogues of natural polymers,such as polylysine, poly(ethylene imine), and the like, can exhibitcytotoxic effects [Lynn & Langer, J. Amer. Chem. Soc., 122:10761-10768(2000)] have lead to the development of alternative synthetic polymersfor tissue engineering applications.

Hydrogels are crosslinked, water-insoluble, water-containing polymerswhich offer good biocompatibility and have a decreased tendency toinduce thrombosis, encrustation, and inflammation and as such are idealcandidates for tissue engineering purposes. The use of hydrogels in cellbiology is well known [see, for example, A. Atala and R. P. Lanza, eds.,“Methods in Tissue Engineering” Academic Press, San Diego, 2002]. A widevariety of hydrogels for in vivo applications have been described [see,for example, the review by Jeong, et al., Adv. Drug Deliv. Rev.,54:37-51 (2002)]. Hydrogels based on N-isopropylacrylamide (NiPAAm) andcertain copolymers thereof, for example, are non-toxic and capable ofsupporting growth of encapsulated cells in vitro [Vernon, et al.,Macromol. Symp., 109:155-167 (1996); Stile, et al., Macromolecules,32:7370-9 (1999); Stile, et al., Biomacromolecules 3: 591- 600.(2002);Stile, et al., Biomacromolecules 2: 185-194.(2001); Webb, et al., MUSCOrthlopaedic J., 3:18-21 (2000); An et al., U.S. Pat. No. 6,103,528].Temperature-sensitive NiPAAm polymers have also been described for usein immunoassays [U.S. Pat. No. 4,780,409]. However, despitemanipulations of synthesis conditions and improvements to enhancebiocompatibility, it is still difficult to obtain a seamlesshost-implant interface and complete integration of the hydrogel implantinto the host [Hicks, et al. Surv. Ophthalmol. 42: 175-189 (1997);Trinkaus-Randall and Nugent, J. Controlled Release 53:205-214 (1998)].

Modifications of synthetic polymer gels with a second naturally derivedpolymer to generate an interpenetrating polymer network (“IPN”)structure have been reported [For example, see Gutowska et al.,Macromolecules, 27:4167 (1994); Yoshida et al., Nature, 374:240 (1995);Wu & Jiang, U.S. Pat. No. 6,030,634; Park et al., U.S. Pat. No.6,271,278]. However, these structures are frequently destabilised byextraction of the naturally derived component by culture media and byphysiological fluids. Naturally derived polymers also tend to biodegraderapidly within the body resulting in destabilisation of in vivoimplants.

More robust hydrogels comprising cross-linked polymer compositions havealso been described. For example, U.S. Pat. No. 6,388,047 describes acomposition consisting of a hydrophobic macromer and a hydrophilicpolymer that are cross-linked to form a hydrogel by free-radicalpolymerisation. U.S. Pat. No. 6,323,278 describes a cross-linked polymercomposition which can form in situ and which comprises two syntheticpolymers, containing multiple electrophilic groups and the othercontaining multiple nucleophilic groups. Both U.S. Pat. No. 6,388,047and 6,384,105 describe systems that must be cross-linked by free radicalchemistry, which requires the use of initiators that are well known tobe cytotoxic (azo compounds, persulfates), thus leading to possible sideeffects if the hydrogel was to be used in the tissue or withencapsulated cells.

U.S. Pat. No. 6,384,105 describes injectable, biodegradable polymercomposites comprising poly(propylene fumarate) and poly(ethyleneglycol)-dimethacrylate which can be cross-linked in situ. The hydrogelsdescribed in this patent are largely based on polymers with apolyethylene oxide backbone polymers. Although these polymers are knownto be biocompatible, their ability to support cell growth is uncertain.

U.S. Pat. No. 6,566,406 describes biocompatible cross-linked hydrogelsthat are formed from water soluble precursors having electrophilic andnucleophilic groups capable of reacting and cross-linking in situ. Theprecursors are described as being a polyalkylene oxide polymer and across-linker. As indicated above, the ability of polyalkylene oxidebackbone polymers to support cell growth is uncertain.

There remains a need therefore, for an improved matrix that isbiocompatible, sufficiently robust to function as an implant and thatcan support cell growth in vivo.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a bio-synthetic matrixand uses thereof. In accordance with an aspect of the present invention,there is provided a synthetic co-polymer suitable for the preparation ofa bio-synthetic matrix, comprising one or more N-alkyl or N,N-dialkylsubstituted acrylamide co-monomer, one or more hydrophilic co-monomerand one or more acryl- or methacryl-carboxylic acid co-monomerderivatised to contain a pendant reactive moiety capable ofcross-linking bioactive molecules, said synthetic polymer having anumber average molecular mass between about 2,000 and about 1,000,000.

In accordance with another aspect of the invention, there is provided abio-synthetic matrix comprising the synthetic co-polymer, a bio-polymerand an aqueous solvent, wherein the synthetic co-polymer and bio-polymerare cross-linked to form a hydrogel.

In accordance with another aspect of the invention, there are provideduses of the bio-synthetic matrix as a scaffold for tissue regeneration,for replacement of damaged or removed tissue in an animal, or forcoating surgical implants.

In accordance with another aspect of the invention, there are providedcompositions comprising: one or more bioactive agent or a plurality ofcells; a synthetic co-polymer of the invention; a bio-polymer; and anaqueous solvent.

In accordance with another aspect of the invention, there is provided animplant for use in tissue engineering comprising a pre-formedbio-synthetic matrix, said matrix comprising an aqueous solvent and abio-polymer cross-linked with a synthetic co-polymer of the invention.

In accordance with another aspect of the invention, there is provided ause of the implant as an artificial cornea.

In accordance with another aspect of the invention, there is provided aprocess for preparing a synthetic co-polymer comprising: (a) dispersingone or more N-alkyl or N,N-dialkyl substituted acrylamide co-monomer,one or more hydrophilic co-monomer and one or more acryl- or methacryl-carboxylic acid co-monomer derivatised to contain a pendantcross-linkable moiety in a solvent in the presence of an initiator; (b)allowing the N-alkyl or N,N-dialkyl substituted acrylamide co-monomer,hydrophilic co-monomer and acryl- or methacryl- carboxylic acidco-monomer to polymerise to form a synthetic co-polymer, and (c)optionally purifying the synthetic co-polymer; and a process forpreparing a bio-synthetic matrix comprising preparing a syntheticco-polymer, dispersing the synthetic co-polymer and a bio-polymer in anaqueous medium and allowing the synthetic co-polymer and the bio-polymerto cross-link to provide the bio-synthetic matrix.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the general structure of a terpolymer according to oneembodiment of the invention comprising N-isopropylacrylamide, (NiPAAm),acrylic acid (AAc) and N-acryloxysuccinimide (ASI).

FIG. 2 presents the clinical results from the transplantation into pigsof artificial corneas prepared from a bio-synthetic matrix according toone embodiment of the invention.

FIG. 3 presents the results of in vivo confocal microscopy at 6 weekspost-operative of artificial corneas prepared from a bio-syntheticmatrix according to one embodiment of the invention and transplantedinto pigs.

FIG. 4 depicts in vivo testing for corneal sensitivity of artificialcorneas prepared from a bio-synthetic matrix according to one embodimentof the invention and transplanted into pigs.

FIGS. 5, 6 and 7 present the results of morphological and biochemicalassessment of artificial corneas prepared from a bio-synthetic matrixaccording to one embodiment of the invention and transplanted into pigs.

FIG. 8 shows (A) the structure of a terpolymer containing a cross-linkedbioactive according to one embodiment of the invention, (B) a cornealscaffold composed of cross-linked collagen and the terpolymer shown in(A) and (C) shows a corneal scaffold composed of thermogelled collagenonly.

FIGS. 9 and 10 depict the results of delivery of a hydrogel containingcollagen and a terpolymer-bioactive agent according to one embodiment ofthe invention into mouse and rat brains.

FIG. 11 shows modulus (A) and stress at failure (B) from suture pull outmeasurements as a function of the concentration ratios ofN-acryloxysuccinimde to collagen amine groups for hydrogel matricesaccording to one embodiment of the invention.

FIG. 12 depicts transmission (A) and back scattering (B) of light acrossthe visible region as a function of the concentration ratios ofN-acryloxysuccinimde to collagen amine groups for hydrogel matricesaccording to one embodiment of the invention.

FIG. 13 depicts transmission (A) and back scattering (B) of light acrossthe visible region as a function of the concentration ratios ofN-acryloxysuccinimde to collagen amine groups for a hydrogel matrixaccording to another embodiment of the invention.

FIG. 14 depicts restoration of touch sensitivity for a pig cornealimplant comprising a hydrogel according to one embodiment of theinvention.

FIG. 15 depicts corneal implantation procedure by lamellar keratoplastyin pigs and clinical in vivo confocal microscopic images of 6-weekimplants comprising a hydrogel according to one embodiment of theinvention. Bar=25 μm for D-F, 15 μm for G-O.

FIG. 16 depicts post-surgical corneal regeneration in pigs receivingcorneal implants comprising a hydrogel according to one embodiment ofthe invention. Bar=100 μm for A-F, 40 μm for G-I, 200 nm for J-L, 20 μmfor M-O, 30 μm for P-R

FIG. 17 depicts implant-host integration post-surgery at 6 weeks postsurgery in pigs receiving corneal implants comprising a hydrogelaccording to one embodiment of the invention. Bar=100 μm in all cases.

FIG. 18 depicts corneal touch sensitivity in implants in pigs receivingcorneal implants comprising a hydrogel according to one embodiment ofthe invention.

FIG. 19 depicts the results of innervation compatibility tests onvarious hydrogel matrices.

FIG. 20 depicts epithelial cell growth and stratification on varioushydrogels. (A) low magnification views of epithelial growth on thehydrogels (inset is higher magnification) and (B) counts of the cellthickness of the epithelium grown over the hydrogels.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular process steps and materials disclosed herein, but is extendedto equivalents thereof as would be recognised by those ordinarilyskilled in the relevant arts. It should also be understood thatterminology employed herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

The term “hydrogel,” as used herein, refers to a cross-linked polymericmaterial which exhibits the ability to swell in water or aqueoussolution without dissolution and to retain a significant portion ofwater or aqueous solution within its structure.

The term “polymer,” as used herein, refers to a molecule consisting ofindividual monomers joined together. In the context of the presentinvention, a polymer may comprise monomers that are joined “end-to-end”to form a linear molecule, or may comprise monomers that are joinedtogether to form a branched structure.

The term “monomer,” as used herein, refers to a simple organic moleculewhich is capable of forming a long chain either alone or in combinationwith other similar organic molecules to yield a polymer.

The term “co-polymer,” as used herein, refers to a polymer thatcomprises two or more different monomers. Co-polymers can be regular,random, block or grafted. A regular co-polymer refers to a co-polymer inwhich the monomers repeat in a regular pattern (e.g. for monomers A andB, a regular co-polymer may have a sequence: ABABABAB). A randomco-polymer is a co-polymer in which the different monomers are arrangedrandomly or statistically in each individual polymer molecule (e.g. formonomers A and B, a random co-polymer may have a sequence:AABABBABBBAAB). In contrast, a block co-polymer is a co-polymer in whichthe different monomers are separated into discrete regions within eachindividual polymer molecule (e.g. for monomers A and B, a blockco-polymer may have a sequence: AAABBBAAABBB). A grafted co-polymerrefers to a co-polymer which is made by linking a polymer or polymers ofone type to another polymer molecule of a different composition.

The term “terpolymer,” as used herein, refers to a co-polymer comprisingthree different monomers.

The term “bio-polymer,” as used herein, refers to a naturally occurringpolymer. Naturally occurring polymers include, but are not limited to,proteins and carbohydrates. The term “bio-polymer” also includesderivatised forms of the naturally occurring polymers that have beenmodified to facilitate cross-linking to a synthetic polymer of theinvention.

The term “synthetic polymer,” as used herein, refers to a polymer thatis not naturally occurring and that is produced by chemical orrecombinant synthesis.

The terms “alkyl” and “lower alkyl” are used interchangeably herein torefer to a straight chain or branched alkyl group of one to eight carbonatoms or a cycloalkyl group of three to eight carbon atoms. These termsare further exemplified by such groups as methyl, ethyl, n-propyl,i-propyl, n-butyl, t-butyl, 1-butyl (or 2-methylpropyl), i-amyl,n-arnyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and thelike.

The term “bioactive agent,” as used herein, refers to a molecule orcompound which exerts a physiological, therapeutic or diagnostic effectin vivo. Bioactive agents may be organic or inorganic. Representativeexamples include proteins, peptides, carbohydrates, nucleic acids andfragments thereof, anti-tumour and anti-neoplastic compounds, anti-viralcompounds, anti-inflammatory compounds, antibiotic compounds such asantifungal and antibacterial compounds, cholesterol lowering drugs,analgesics, contrast agents for medical diagnostic imaging, enzymes,cytokines, local anaesthetics, hormones, anti-angiogenic agents,neurotransmitters, therapeutic oligonucleotides, viral particles,vectors, growth factors, retinoids, cell adhesion factors, extracellularmatrix glycoproteins (such as laminin), hormones, osteogenic factors,antibodies and antigens.

The term “biocompatible,” as used herein, refers to an ability to beincorporated into a biological system, such as into an organ or tissueof an animal, without stimulating an immune and/or inflammatoryresponse, fibrosis or other adverse tissue response.

As used herein, the term “about” refers to a ±10% variation from thenominal value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

1. Bio-Syntheticmatrix

The present invention provides a bio-synthetic matrix comprising ahydrogel which is formed by cross-linking a synthetic polymer and abio-polymer. The bio-polymer may be in its naturally-occurring form, orit may be derivatised to facilitate cross-linking to the syntheticpolymer. The matrix is robust, biocompatible and non-cytotoxic. Thematrix can be formed in aqueous solution at neutral pH and can betailored to further comprise one or more bioactive agents such as growthfactors, retinoids, cell adhesion factors, enzymes, peptides, proteins,nucleotides, drugs, and the like. The bioactive agent can be covalentlyattached to the synthetic polymer, or it may be encapsulated anddispersed within the final matrix depending on the end use demands forthe matrix. The matrix may also comprise cells encapsulated anddispersed therein, which are capable of proliferation and/ordiversification upon deposition of the matrix in vivo.

In one embodiment of the present invention, the bio-synthetic matrixsupports cell growth. Such cell growth may be epithelial and/orendothelial surface coverage (i.e. two dimensional, 2D, growth) and/orthree-dimensional (3D) cell growth involving growth into the matrixitself.

In another embodiment of the invention, the bio-synthetic matrixsupports nerve in-growth. As is known in the art, nerve growth intotransplanted tissue takes place over an extended period of time,typically in the order of months or years. Growth of nerves into thematrix can occur more rapidly than growth of nerves into transplantedtissue thus leading to more rapid regeneration of functional tissue, forexample, nerve in-growth may occur within weeks.

The bio-synthetic matrix can be tailored for specific applications. Forexample, the matrix can be used in tissue engineering applications andmay be pre-formed into a specific shape for this purpose. The matrix canalso be used as a drug delivery vehicle to provide sustained release ofa therapeutic or diagnostic compound at a particular site within thebody of an animal.

In order to be suitable for in vivo implantation for tissue engineeringpurposes, the bio-synthetic matrix must maintain its form atphysiological temperatures, be substantially insoluble in water, beadequately robust, and support the growth of cells. It may also bedesirable for the matrix to support the growth of nerves.

1.1 Synthetic Polymer

In accordance with the present invention, the synthetic polymer that isincorporated into the bio-synthetic matrix is a co-polymer comprisingone or more acrylamide derivatives, one or more hydrophilic co-monomersand one or more derivatised carboxylic acid co-monomers which comprisependant cross-linkable moieties.

As used herein, an “acrylamide derivative” refers to a N-alkyl orN,N-dialkyl substituted acrylamide or methacrylamide. Examples ofacrylamide derivatives suitable for use in the synthetic polymer of thepresent invention include, but are not limited to, N-methylacrylamide,N-ethylacrylamide, N-isopropylacrylamide (NiPAAm), N-octylacrylamide,N-cyclohexylacrylamide, N-methyl-N-ethylacrylamide,N-methylmethacrylamide, N-ethylmethacrylamide,N-isopropylmethacrylamide, N,N-dimethylacrylamide,N,N-diethylacrylamide, N,N-dimethylmethacrylamide,N,N-diethylmethacrylamide, N,N-dicyclohexylacrylamide,N-methyl-N-cyclohexylacrylamide, N-acryloylpyrrolidine,N-vinyl-2-pyrrollidinone, N-methacryloylpyrrolidine, and combinationsthereof.

A “hydrophilic co-monomer” in the context of the present invention is ahydrophilic monomer that is capable of co-polymerisation with theacrylamide derivative and the derivatised carboxylic acid components ofthe synthetic polymer. The hydrophilic co-monomer is selected to provideadequate solubility for polymerisation, aqueous solubility of theco-polymer and freedom from phase transition of the final co-polymer andhydrogel. Examples of suitable hydrophilic co-monomers are hydrophilicacryl- or methacryl-compounds such as carboxylic acids including acrylicacid, methacrylic acid and derivatives thereof, acrylamide,methacrylamide, hydrophilic acrylamide derivatives, hydrophilicmethacrylamide derivatives, hydrophilic acrylic acid esters, hydrophilicmethacrylic acid esters, vinyl ethanol and its derivatives and ethyleneglycols. The carboxylic acids and derivatives may be, for example,acrylic acid, methacrylic acid, 2-hydroxyethyl methacrylate (HEMA), or acombination thereof Examples of hydrophilic acrylamide derivativesinclude, but are not limited to, N,N-dimethylacrylamide,N,N-diethylacrylamide, 2-[N,N-dimethylamino]ethylacrylamide,2-[N,N-diethylamino]ethylacrylamide, N,N-diethylmethacrylamide,2-[N,N-dimethylamino]ethylmethacrylamide,2-[N,N-diethylamino]ethylmethacrylamide, N-vinyl-2-pyrrollidinone, orcombinations thereof. Examples of hydrophilic acrylic esters include,but are not limited to, 2-[N,N-diethylamino]ethylacrylate,2-[N,N-dimethylamino]ethylacrylate,2-[N,N-diethylamino]ethylmethacrylate,2-[N,N-dimethylamino]ethylmethacrylate, or combinations thereof.

As used herein, a “derivatised carboxylic acid co-monomer” refers to ahydrophilic acryl- or methacryl- carboxylic acid, for example, acrylicacid, methacrylic acid, or a substituted version thereof, which has beenchemically derivatised to contain one or more cross-linking moieties,such as succinimidyl groups, imidazoles, benzotriazoles andp-nitrophenols. The term “succinimidyl group” is intended to encompassvariations of the generic succinimidyl group, such as sulphosuccinimidylgroups. Other similar structures such as 2-(N-morpholino)ethanesulphonicacid will also be apparent to those skilled in the art. In the contextof the present invention the group selected as a cross-linking moietyacts to increase the reactivity of the carboxylic acid group to which itis attached towards primary amines (i.e. —NH₂ groups) and thiols (i.e.—SH groups). Examples of suitable groups for derivatisation of thecarboxylic acid co-monomers for use in the synthetic polymer include,but are not limited to, N-succinimide, N-succinimide-3-sulphonic acid,N-benzotriazole, N-imidazole and p-nitrophenol.

In one embodiment of the present invention, the synthetic polymercomprises:

(a) one or more acrylamide derivative of general formula I:

-   -   wherein:    -   R₁, R₂, R₃, R₄ and R₅ are independently selected from the group        of: H and lower alkyl;

(b) one or more hydrophilic co-monomer (which may be the same ordifferent to (a)) having the general formula II:

-   -   wherein:

Y is O or is absent;

-   -   R₆, and R₇ are independently selected from the group of: H and        lower alkyl;    -   R₈ is H, lower alkyl, or —OR′, where R′ is H or lower alkyl; and    -   R₉ is H, lower alkyl, or —C(O)R₁₀, and    -   R₁₀ is —NR₄R₅ or —OR″, where R″ is H or CH₂CH₂OH;    -   and (c) one or more derivatised carboxylic acid having the        general formula III:

wherein:

-   -   R₁₁, R₁₂ and R₁₃ are independently selected from the group of: H        and lower alkyl and    -   Q is N-succinimido, 3-sulpho-succinimdo (sodium salt),        N-benzotriazolyl, N-imidazolyl orp-nitrophenyl.

In one embodiment, the synthetic polymer comprises one or moreacrylamide derivative of general formula L one or more hydrophilicco-monomer of general formula II and one or more derivatised carboxylicacid of general formula III, as described above, wherein the term “loweralkyl” refers to a branched or straight chain alkyl group having 1 to 8carbon atoms.

In another embodiment, the synthetic polymer comprises one or moreacrylamide derivative of general formula I, one or more hydrophilicco-monomer of general formula II and one or more derivatised carboxylicacid of general formula III, as described above, wherein the term “loweralkyl” refers to to a cycloalkyl group having 3 to 8 carbon atoms, suchas cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The co-polymer may be linear or branched, regular, random or block. Inaccordance with the present invention, the final synthetic polymercomprises a plurality of pendant reactive moieties available forcross-linking, or grafting, of appropriate biomolecules.

A worker skilled in the art will appreciate that the group of compoundsencompassed by the term “acrylamide derivative” and the group ofcompounds encompassed by the term “hydrophilic co-monomer” overlapsubstantially and that a single monomer could be selected that fulfilsthe functions of both these components in the co-polymer. Thus, forexample, when an acrylamide derivative is selected that is sufficientlyhydrophilic to confer on the synthetic polymer the desired properties,then a hydrophilic co-monomer component may be chosen that is identicalto the selected acrylamide derivative resulting in a co-polymer thatcomprises two different monomers only (i.e. the acrylamidederivative/hydrophilic co-monomer and the derivatised carboxylic acidco-monomer). On the other hand, when enhanced hydrophilicity beyond thatprovided by the selected acrylamide derivative is desired, then one ormore different hydrophilic co-monomers may be chosen resulting in aco-polymer comprising at least three different monomers.

In one embodiment of the present invention, the acrylamide derivativeand the hydrophilic co-monomer used in the preparation of the syntheticpolymer are the same. In another embodiment, the acrylamide derivativeand the hydrophilic co-monomer used in the preparation of the syntheticpolymer are different.

The overall hydrophilicity of the co-polymer is controlled to conferwater solubility at 0° C. to physiological temperatures withoutprecipitation or phase transition. In one embodiment of the presentinvention, the co-polymer is water soluble between about 0° C. and about37° C.

The co-polymer should be sufficiently soluble in aqueous solution tofacilitate hydrogel formation. In accordance with one embodiment of thepresent invention, therefore, the term “water soluble” is intended torefer to an aqueous solubility of the co-polymer of at least about 0.5weight/volume (w/v) %. In another embodiment, the co-polymer has anaqueous solubility of between about 1.0 w/v % and about 50 w/v %. In afurther embodiments, the co-polymer has an aqueous solubility of about 5w/v % and about 45 w/v % and between about 10% w/v and about 35% w/v.

As is known in the art, most synthetic polymers have a distribution ofmolecular mass and various different averages of the molecular mass areoften distinguished, for example, the number average molecular mass(M_(n)) and the weight average molecular mass (M_(w)). The molecularweight of a synthetic polymer is usually defined in terms of its numberaverage molecular mass (M_(n)), which in turn is defined as the sum ofn_(i)M_(i) divided by the sum of n_(i), where n_(i) is the number ofmolecules in the distribution with mass M_(i). The synthetic polymer foruse in the matrix of the present invention typically has a numberaverage molecular mass (M_(n)) between 2,000 and 1,000,000. In oneembodiment of the present invention, the M_(n) of the polymer is betweenabout 5,000 and about 90,000. In another embodiment of the presentinvention, the M_(n) of the polymer is between about 25,000 and about80,000. In a further embodiment, the M_(n) of the polymer is betweenabout 30,000 and about 50,000. In another embodiment, the M_(n) of thepolymer is between about 50,000 and about 60,000.

It is also well-known in the art that certain water-soluble polymersexhibit a lower critical solution temperature (LCST) or “cloud point.”The LCST of a polymer is the temperature at which phase separationoccurs (i.e. the polymer begins to separate from the surrounding aqueousmedium). Typically, for those polymers or hydrogels that are clear, theLCST also corresponds to the point at which clarity begins to be lost.It will be readily apparent that for certain tissue engineeringapplications, the presence or absence of phase separation in the finalhydrogel may not be relevant provided that the hydrogel still supportscell growth. For other applications, however, a lack of phase separationin the final hydrogel may be critical. For example, for opticalapplications, clarity (and, therefore, the absence of any phasetransition) will be important.

Thus, in accordance with one embodiment of the present invention,co-polymers with a LCST between about 35° C. and about 60° C. areselected for use in the hydrogels. In another embodiment, co-polymerswith a LCST between about 42° C. and about 60° C. are selected for usein the hydrogels It is also known in the art that the LCST of a polymermay be affected by the presence of various solutes, such as ions orproteins, and by the nature of compounds cross-linked or attached to thepolymer. Such effects can be determined empirically using standardtechniques and selection of a synthetic polymer with an appropriate LCSTfor a particular application is considered to be within the ordinaryskills of a worker in the art.

In order for the synthetic polymer to be suitably robust andthermostable, it is important that the ratio of acrylamide derivative(s)to hydrophilic co-monomer(s) is optimised when different monomers areused for these components. Accordingly, the acrylamide derivative(s) arepresent in the synthetic polymer in the highest molar ratio. Inaddition, the number of derivatised carboxylic acid co-monomer(s)present in the final polymer will determine the ability of the syntheticgel to form cross-links with the bio-polymer in the bio-syntheticmatrix. Selection of suitable molar ratios of each component to providea final synthetic polymer with the desired properties is within theordinary skills of a worker in the art.

In accordance with one embodiment of the present invention, whendifferent monomers are being used as the acrylamide derivative andhydrophilic co-monomer components, the amount of acrylamide derivativein the polymer is between 50% and 90%, the amount of hydrophilicco-monomer is between 5% and 50%, and the amount of derivatisedcarboxylic acid co-monomer is between 0.1% and 15%, wherein the sum ofthe amounts of acrylamide derivative, hydrophilic co-monomer andderivatised carboxylic acid co-monomer is 100%, wherein the % valuerepresents the molar ratio.

In accordance with another embodiment of the invention, the syntheticpolymer is prepared using the same monomer as both the acrylamidederivative and the hydrophilic co-monomer and the molar ratio of theacrylamide derivative/hydrophilic co-monomer is between about 50% andabout 99.5% and the molar ratio of the derivatised carboxylic acidco-monomer is between about 0.5% and about 50%.

In accordance with a further embodiment, the combined molar ratio of theacrylamide derivative and the hydrophilic co-monomer is between about80% and about 99% and the molar ratio of the derivatised carboxylic acidco-monomer is between about 1% and about 20%.

One skilled in the art will appreciate that the selection and ratio ofthe components in the synthetic polymer will be dependent to varyingdegrees on the final application for the bio-synthetic matrix. Forexample, for ophthalmic applications, it is important that the finalmatrix be clear, whereas for other tissue engineering applications, theclarity of the matrix may not be an important factor.

In one embodiment of the present invention, the synthetic polymer is arandom or block co-polymer comprising one acrylamide derivative, onehydrophilic co-monomer and one derivatised carboxylic acid co-monomer (a“terpolymer”). In another embodiment, the synthetic polymer is aterpolymer comprising NiPAAm monomer, acrylic acid (AAc) monomer and aderivatised acrylic acid monomer. In a further embodiment, the syntheticpolymer is a terpolymer comprising NiPAAm monomer, acrylamide (AAm)monomer and derivatised acrylic acid monomer. In another embodiment, thederivatised acrylic acid monomer is N-acryloxysuccinimide. In anotherembodiment, a terpolymer is prepared with a feed ratio that comprisesNiPAAm monomer, AAc monomer and N-acryloxysuccinimide in a ratio ofabout 85:10:5 molar %.

In an alternate embodiment of the invention, the synthetic polymer is arandom or block co-polymer comprising one acrylamidederivative/hydrophilic co-monomer and one carboxylic acid co-monomer. Inanother embodiment, the synthetic polymer comprises DMAA monomer and aderivatised acrylic acid monomer. In a further embodiment, thederivatised acrylic acid monomer is N-acryloxysuccinimide. In anotherembodiment, a synthetic polymer is prepared with a feed ratio thatcomprises DMAA monomer and N-acryloxysuccinimide in a ratio of about95:5 molar %.

1.2 Bio-Polymer

Bio-polymers are naturally-occurring polymers, such as proteins andcarbohydrates. In accordance with the present invention, thebio-synthetic matrix comprises a bio-polymer or a derivatised versionthereof cross-linked to the synthetic polymer by means of the pendantcross-linking moieties in the latter. Thus, for the purposes of thepresent invention the bio-polymer contains one or more groups which arecapable of reacting with the cross-linking moiety (e.g. a primary amineor a thiol), or can be derivatised to contain such a group. Examples ofsuitable bio-polymers for use in the present invention include, but arenot limited to, collagens (including Types I, II, III, IV and V),denatured collagens (or gelatins), recombinant collagens,fibrin-fibrinogen, elastin, glycoproteins, alginate, chitosan,hyaluronic acid, chondroitin sulphates and glycosaminoglycans (orproteoglycans). One skilled in the art will appreciate that some ofthese bio-polymers may need to be derivatised in order to contain asuitable reactive group as indicated above, for example,glucosaminoglycans need to be deacetylated or desulphated in order topossess a primary amine group. Such derivatisation can be achieved bystandard techniques and is considered to be within the ordinary skillsof a worker in the art.

Suitable bio-polymers for use in the invention can be purchased fromvarious commercial sources or can be prepared from natural sources bystandard techniques.

1.3 Bioactive Agents

As indicated above, the synthetic polymer to be included in thebio-synthetic matrix the present invention contains a plurality ofpendant cross-linking moieties. It will be apparent that sufficientcross-linking of the synthetic and bio-polymers to achieve a suitablyrobust matrix can be achieved without reaction of all free cross-linkinggroups. Excess groups may, therefore, optionally be used to covalentlyattach desirable bioactive agents to the matrix. Non-limiting examplesof bioactive agents that may be incorporated into the matrix bycross-linking include, for example, growth factors, retinoids, enzymes,cell adhesion factors, extracellular matrix glycoproteins (such aslaminin, fibronectin, tenascin and the like), hormones, osteogenicfactors, cytokines, antibodies, antigens, and other biologically activeproteins, certain pharmaceutical compounds, as well as peptides,fragments or motifs derived from biologically active proteins.

In one embodiment of the present invention, the cross-linking groups aresuccinimidyl groups and suitable bioactive agents for grafting to thepolymer are those which contain either primary amino or thiol groups, orwhich can be readily derivatised so as to contain these groups.

2. Method of Preparing the Bio-Synthetic Matrix

2.1 Preparation of the Synthetic Polymer

Co-polymerization of the components for the synthetic polymer can beachieved using standard methods known in the art [for example, see A.Ravve “Principles of Polymer Chemistry”, Chapter 3. Plenum Press, NewYork 1995]. Typically appropriate quantities of each of the monomers aredispersed in a suitable solvent in the presence of an initiator. Themixture is maintained at an appropriate temperature and theco-polymerisation reaction is allowed to proceed for a pre-determinedperiod of time. The resulting polymer can then be purified from themixture by conventional methods, for example, by precipitation.

The solvent for the co-polymerisation reaction may be a non-aqueoussolvent if one or more monomer is sensitive to hydrolysis or it may bean aqueous solvent. Suitable aqueous solvents include, but are notlimited to, water, buffers and salt solutions. Suitable non-aqueoussolvents are typically cyclic ethers (such as dioxane), chlorinatedhydrocarbons (for example, chloroform) or aromatic hydrocarbons (forexample, benzene). The solvent may be nitrogen purged prior to use, ifdesired. In one embodiment of the present invention, the solvent is anon-aqueous solvent. In another embodiment, the solvent is dioxane.

Suitable polymerisation initiators are known in the art and are usuallyfree-radical initiators. Examples of suitable initiators include, butare not limited to, 2,2′-azobisisobutyronitrile (AIBN), other azocompounds, such as 2,2′-azobis-2-ethylpropionitrile;2,2′-azobis-2-cyclopropylpropionitrile; 2,2′-azobiscyclohexanenitrile;2,2′-azobiscyclooctanenitrile, and peroxide compounds, such as dibenzoylperoxide and its substituted analogues, and persulfates, such as sodium,potassium, and the like.

Once the polymer has been prepared, and purified if necessary, it can becharacterised by various standard techniques. For example, the molarratio composition of the polymer can be determined by nuclear magneticresonance spectroscopy (proton and/or carbon-13) and bond structure canbe determined by infrared spectroscopy. Molecular mass can be determinedby gel permeation chromatography and/or high pressure liquidchromatography. Thermal characterisation of the polymer can also beconducted, if desired, for example by determination of the melting pointand glass transition temperatures using differential scanningcalorimetric analysis. Aqueous solution properties such as micelle andgel formation, and LCST can be determined using visual observation,fluorescence spectroscopy, UV-visible spectroscopy and laser lightscattering instruments.

In one embodiment of the present invention, the synthetic polymer isprepared by dispersing the monomers in nitrogen-purged dioxane in thepresence of the initiator AIBN and allowing polymerisation to proceed ata temperature of about 60° C. to 70° C. The resulting polymer ispurified by repeated precipitation.

2.2 Preparation of the Hydrogel

Cross-linking of the synthetic polymer and bio-polymer can be readilyachieved by mixing appropriate amounts of each polymer at roomtemperature in a suitable solvent. Typically the solvent is an aqueoussolvent, such as a salt solution, buffer solution, cell culture medium,or a diluted or modified version thereof. One skilled in the art willappreciate that in order to preserve triple helix structure of polymerssuch as collagen and to prevent fibrillogenisis and/or opacification ofthe hydrogel, the cross-linking reaction should be conducted in aqueousmedia with close control of the pH and temperature. The significantlevels of amino acids in nutrient media normally used for cell culturecan cause side reactions with the cross-linking moieties of thesynthetic polymer, which can result in diversion of these groups fromthe cross-linking reaction. Use of a medium free of amino acids andother proteinaceous materials can help to prevent these side reactionsand, therefore, increase the number of cross-links that form between thesynthetic and bio-polymers. Conducting the cross-linking reaction inaqueous solution at room or physiological temperatures allows bothcross-linking and the much slower hydrolysis of any residualcross-linking groups to take place.

Alternatively, a termination step can be included to react any residualcross-liking groups in the matrix. For example, one or more wash stepsin a suitable buffer containing glycine will terminate any residualcross-linking groups as well as removing any side products generatedduring the cross-linking reaction. Unreacted cross-linking groups mayalso be terminated with a polyfunctional amine such as lysine ortriethylenetetraamine leading to formation of additional short,inter-chain cross-links. Wash steps using buffer alone can also beconducted if desired in order to remove any side products from thecross-linking reaction. If necessary, after the cross-linking step, thetemperature of the cross-linked polymer suspension can be raised toallow the hydrogel to form fully.

In accordance with the present invention, the components of the hydrogelare chemically cross-linked so as to be substantially non-extractable,i.e. the bio-polymer and synthetic polymer do not exude extensively fromthe gel under physiological conditions. In accordance with oneembodiment of the present invention, the amount of bio-polymer orsynthetic polymer that can be extracted from the matrix into aqueousmedia under physiological conditions over a period of 24 hours is lessthan 5% by weight of either component. In another embodiment, the amountof bio-polymer or synthetic polymer that can be extracted from thematrix into aqueous media under physiological conditions over a periodof 24 hours is less than 2% by weight of either component. In furtherembodiments, the amount that can be extracted over a period of 24 hoursis less than 1% by weight, less than 0.5% by weight and less than 0.2%by weight.

The amount of bio-polymer and/or synthetic polymer that can be extractedfrom the matrix into aqueous media can be determined in vitro usingstandard techniques (for example, the USP Basket Method). Typically, thematrix is placed in an aqueous solution of a predetermined pH, forexample around pH 7.4 to simulate physiological conditions. Thesuspension may or may not be stirred. Samples of the aqueous solutionare removed at predetermined time intervals and are assayed for polymercontent by standard analytical techniques.

One skilled in the art will understand that the amount of each polymerto be included in the hydrogel will be dependent on the choice ofpolymers and the intended application for the hydrogel. In general,using higher initial amounts of each polymer will result in theformation of a more robust gel due to the lower water content and thepresence of a greater amount of cross-linked polymer. Higher quantitiesof water or aqueous solvent will produce a soft hydrogel. In accordancewith the present invention, the final hydrogel comprises between about20 and 99.6% by weight of water or aqueous solvent, between about 0.1and 30% by weight of synthetic polymer and between about 0.3 and 50% byweight of bio-polymer.

In one embodiment of the present invention, the final hydrogel comprisesbetween about 40 and 99.6% by weight of water or aqueous solvent,between about 0.1 and 30% by weight of synthetic polymer and betweenabout 0.3 and 30% by weight of bio-polymer. In another embodiment, thefinal hydrogel comprises between about 60 and 99.6% by weight of wateror aqueous solvent, between about 0.1 and 10% by weight of syntheticpolymer and between about 0.3 and 30% by weight of bio-polymer. In afurther embodiment, the final hydrogel comprises between about 80 and98.5% by weight of water or aqueous solvent, between about 0.5 and 5% byweight of synthetic polymer and between about 1 and 15% by weight ofbio-polymer. In other embodiments, the final hydrogel contains about 95to 97% by weight of water or aqueous solvent and between about 1-2% byweight of synthetic polymer and about 2-3% by weight of bio-polymer; andabout 94 to 98% by weight of water or aqueous solvent and between about1-3% by weight of synthetic polymer and about 1-3% by weight ofbio-polymer.

Similarly, the relative amounts of each polymer to be included in thehydrogel will be dependent on the type of synthetic polymer andbio-polymer being used and upon the intended application for thehydrogel. One skilled in the art will appreciate that the relativeamounts bio-polymer and synthetic polymer will influence the final gelproperties in various ways, for example, high quantities of bio-polymerwill produce a very stiff hydrogel. One skilled in the art willappreciate that the relative amounts of each polymer in the final matrixcan be described in terms of the weight : weight ratio of thebio-polymer: synthetic polymer or in terms of equivalents of reactivegroups. In accordance with the present invention, the weight per weightratio of bio-polymer: synthetic polymer is between about 1:0.07 andabout 1:14. In one embodiment, the w/w ratio of bio-polymer: syntheticpolymer is between 1:1.3 and 1:7. In another embodiment, the w/w ratioof bio-polymer: synthetic polymer is between 1:1 and 1:3. In a furtherembodiment, the w/w ratio of bio-polymer: synthetic polymer is between1:0.7 and 1:2.

In an alternative embodiment of the present invention, the matrixcomprises a proteinaceous bio-polymer and a synthetic polymer comprisingpendant N-acryloxysuccinimide groups. In this embodiment of theinvention, the ratio of bio-polymer: synthetic polymer is described interms of molar equivalents of free amine groups in the bio-polymer toN-acryloxysuccinimide groups and is between 1:0.5 and 1:20. In anotherembodiment, this ratio is between 1:1.8 and 1:10. In a furtherembodiments, the ratio is between 1:1 and 1:5, and between 1:1 and 1:3.

2.3 Incorporation of Bioactive Agents into the Bio-Synthetic Matrix

Bioactive agents can be incorporated into the matrix if desired eitherby covalent attachment (or “grafting”) to the synthetic polymer throughthe pendant cross-linking moieties, or by encapsulation within thematrix. Examples of bioactive agents that may be covalently attached tothe synthetic polymer component of the matrix are provided above. Ifnecessary, the bioactive agent may be first derivatised by standardprocedures to provide appropriate reactive groups for reaction with thecross-linking groups. For covalent attachment of a bioactive agent, thesynthetic polymer is first reacted with the bioactive agent and thenthis modified synthetic polymer subsequently cross-linked to thebio-polymer as described above. Reaction of the bioactive agent with thesynthetic polymer can be conducted under standard conditions, forexample by mixing the bioactive agent and the synthetic polymer togetherin a non-aqueous solvent, such as N,N-dimethyl formamide, dioxane,dimethyl sulphoxide and N,N-dimethylacrylamide. The use of a non-aqueoussolvent avoids hydrolysis of the reactive groups during incorporation ofthe bioactive agent. Alternatively, the reaction may be conducted in anaqueous solvent as described above for the cross-linking reaction.

Bioactive agents which are not suitable for grafting to the polymer, forexample, those that do not contain primary amino or free thiol groupsfor reaction with the cross-linking groups in the synthetic polymer, orwhich cannot be derivatised to provide such groups, can be entrapped inthe final matrix. Examples of bioactive agents which may be entrapped inthe matrix include, but are not limited to, pharmaceutical drugs,diagnostic agents, viral vectors, nucleic acids and the like. Forentrapment, the bioactive agent is added to a solution of the syntheticpolymer in an appropriate solvent prior to mixture of the syntheticpolymer and the bio-polymer to form a cross-linked hydrogel.Alternatively, the bioactive agent can be added to a solution containingboth the synthetic and bio-polymers prior to the cross-linking step. Thebioactive agent is mixed into the polymer solution such that it issubstantially uniformly dispersed therein, and the hydrogel issubsequently formed as described above. Appropriate solvents for usewith the bioactive agent will be dependent on the properties of theagent and can be readily determined by one skilled in the art.

2.4 Entrapment of Cells in the Bio-Synthetic Matrix

The bio-synthetic matrix according to the present invention may alsocomprise cells entrapped therein and thus permit delivery of the cellsto a tissue or organ in vivo. A variety of different cell types may bedelivered using the bio-synthetic matrix, for example, myocytes, ocularcells (e.g. from the different corneal layers), adipocytes, 30fibromyoblasts, ectodermal cells, muscle cells, osteoblasts (i.e. bonecells), chondrocytes (i.e. cartilage cells), endothelial cells,fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrowcells, neural cells, genitourinary cells (including nephritic cells), orcombinations thereof. The matrix may also be used to deliver totipotentstem cells, pluripotent or committed progenitor cells or re-programmed(dedifferentiated) cells to an in vivo site such that cells of the sametype as the tissue can be produced. For example, mesenchymal stem cells,which are undifferentiated, can be delivered in the matrix. Examples ofmesenchymal stem cells include those which can diversify to produceosteoblasts (to generate new bone tissue), chondrocytes (to generate newcartilaginous tissue), and fibroblasts (to produce new connectivetissue). Alternatively, committed progenitor cells capable ofproliferating to provide cells of the same type as those present at thein vivo site can be used, for example, myoblasts, osteoblasts,fibroblasts and the like.

Cells can be readily entrapped in the final matrix by addition of thecells to a solution of the synthetic polymer prior to admixture with thebio-polymer to form a cross-linked hydrogel. Alternatively, the cellscan be added to a solution containing both the synthetic andbio-polymers prior to the cross-linking step. The synthetic polymer maybe reacted with a bioactive agent prior to admixture with the cells ifdesired. Typically, for the encapsulation of cells in the matrix, thevarious components (cells, synthetic polymer and bio-polymer) aredispersed in an aqueous medium, such as a cell culture medium or adiluted or modified version thereof. The cell suspension is mixed gentlyinto the polymer solution until the cells are substantially uniformlydispersed in the solution, then the hydrogel is formed as describedabove.

2.5 Other Elements

The present invention also contemplates the optional inclusion of one ormore reinforcing material in the bio-synthetic matrix to improve themechanical properties of the matrix such as the strength, resilience,flexibility and/or tear resistance. Thus, the matrix may be reinforcedwith flexible or rigid fibres, fibre mesh, fibre cloth and the like. Theuse of such reinforcing materials is known in the art, for example, theuse of fibres, cloth, or sheets made from collagen fibrils, oxidisedcellulose or polymers such as polylactic acid, polyglycolic acid orpolytetrafluoroethylene in implantable medical applications is known.

The reinforcing material can be incorporated into the matrix usingstandard protocols. For example, an aqueous solution of synthetic andbio-polymers in an appropriate buffer can be added to a fibre cloth ormesh, such as Interceed (Ethicon Inc., New Brunswick, N.J.). The aqueoussolution will flow into the interstices of the cloth or mesh prior toundergoing cross-linking and will thus form a hydrogel with the cloth ormesh embedded therein. Appropriate moulds can be used to ensure that thefibres or fibre mesh are contained entirely within the hydrogel ifdesired. The composite structure can subsequently be washed to removeany side products generated during the cross-linking reaction.Typically, the fibres used are hydrophilic in nature to ensure completewetting by the aqueous solution of polymers.

One skilled in the art will appreciate that, for applications requiringhigh optical clarity, the structure of the reinforcement should beselected to prevent light scattering from the final composite matrix,for example, by the use of nano-fibers and/or careful refractive indexmatching of reinforcement and hydrogel.

3. Testing the Biosynthetic Matrix

In accordance with the present invention, the bio-synthetic matrixcomprises a hydrogel with or without added bioactive agents and/orencapsulated cells. In order to be suitable for in vivo implantation fortissue engineering purposes, the bio-synthetic matrix must maintain itsform at physiological temperatures, be adequately robust, besubstantially insoluble in water, and support the growth of cells. Itmay also be desirable for the matrix to support the growth of nerves. Itwill be readily appreciated that for certain specialised applications,the matrix may require other characteristics. For example, for surgicalpurposes, the matrix may need to be relatively flexible as well asstrong enough to support surgical manipulation with suture thread andneedle, and for ophthalmic applications, such as cornea repair orreplacement, the optical clarity of the matrix will be important.

3.1 Physical/Chemical Testing

When used for tissue engineering applications, the bio-synthetic matrixneeds to meet the mechanical parameters necessary to prevent the matrixtearing or rupturing when submitted to surgical procedures and toprovide adequate support for cell growth once in place. The ability ofmatrix to resist tearing is related to its intrinsic mechanicalstrength, the form and thickness of the matrix and the tension beingapplied. The ability of the bio-synthetic matrix to withstand shearingforces, or tearing can be roughly determined by applying forces inopposite directions to the specimen using two pairs of forceps.Alternatively, a suitable apparatus can be used to measurequantitatively the ability of the matrix to withstand shearing forces.Tensiometers for this purpose are available commercially, for example,from MTS, Instron, and Cole Parmer.

For testing, the matrix can be formed into sheets and then cut intoappropriately sized strips. Alternatively, the matrix can be mouldedinto the desired shape for tissue engineering purposes and the entiremoulded matrix can be tested. To calculate tensile strength, the forceat rupture, or “failure,” of the matrix is divided by thecross-sectional area of the test sample, resulting in a value that canbe expressed in force (N) per unit area. The stiffness (modulus) of thematrix is calculated from the slope of the linear portion of thestress/strain curve. Strain is the real-time change in length during thetest divided by the initial length of the test sample before the testbegins. The strength at rupture is the final length of the test samplewhen it ruptures minus the length of the initial test sample, divided bythis initial length.

One skilled in the art will appreciate that because of the softness ofhydrogels and exudation of the aqueous component when clamped,meaningful tensile data can be difficult to obtain from hydrogels.Quantitative characterisation of tensile strength in hydrogels can beachieved, for example, through the use of suture pull-out measurementson moulded matrix samples. Typically, a suture is placed about 2 mm fromthe edge of a test sample and the peak force that needs to be applied inorder to rip the suture through the sample is measured. For example, fora test sample of matrix intended for ophthalmic applications that hasbeen moulded in the shape and thickness of a human cornea, twodiametrically opposed sutures can be inserted into the matrix, as wouldbe required for the first step in ocular implantation. The two suturescan then be pulled apart at about 10 mm/min on a suitable instrument,such as an Instron Tensile Tester. Strength at rupture of the matrix iscalculated, together with elongation at break and elastic modulus [see,for example, Zeng et al., J. Biomech., 34:533-537 (2001)]. It will beappreciated by those skilled in the art that, for those bio-syntheticmatrices intended for surgical applications, the matrices need not be asstrong (i.e. have the same ability to resist tearing) as mammaliantissue. The determining factor for the strength of the matrix in suchapplications is whether or not it can be sutured in place by a carefuland experienced surgeon.

If desired, the LCST of the bio-synthetic hydrogel matrix can bemeasured using standard techniques. For example, LCST can be calculatedby heating samples of the matrix at about 0.2° C. per minute andvisually observing the cloud point (see, for example, H. Uludag, et al.,J. Appl. Polym. Sci. 75:583-592 (2000)).

Permeability of the bio-synthetic matrix can be determined by assessingthe glucose permability coefficient and/or the average pore sizes forthe matrix using standard techniques such as PBS permeability assessmentusing a permeability cell and/or atomic force microscopy. In accordancewith one embodiment of the present invention, the bio-synthetic matrixhas an average pore size between about 90 nm and about 500 nm. Inanother embodiment, the matrix has an average pore size between about100 nm and about 300 nm.

Optical transmission and light scatter can also be measured for matricesintended for ophthalmic applications using a custom-built instrumentthat measures both transmission and scatter [see, for example, Priestand Munger, Invest. Ophthalmol. Vis. Sci. 39: S352 (1998)].

3.2 In Vitro Testing

It will be readily appreciated that the bio-synthetic matrix must benon-cytotoxic and biocompatible in order to be suitable for in vivo use.The cytotoxicity of the bio-synthetic matrix can be assessed usingstandard techniques such as the Ames assay to screen for mutagenicactivity, the mouse lymphoma assay to screen for the ability of thematrix to induce gene mutation in a mammalian cell line, in vitrochromosomal aberration assays using, for example, Chinese hamster ovarycells (CHO) to screen for any DNA rearrangements or damage induced bythe matrix. Other assays include the sister chromatid assay, whichdetermines any exchange between the arms of a chromosome induced by thematrix and in vitro mouse micronucleus assays to determine any damage tochromosomes or to the mitotic spindle. Protocols for these and otherstandard assays are known in the art, for example, see OECD Guidelinesfor the Testing of Chemicals and protocols developed by the ISO.

The ability of the matrix to support cell growth can also be assessed invitro using standard techniques. For example, cells from an appropriatecell line, such as human epithelial cells, can be seeded either directlyonto the matrix or onto an appropriate material surrounding the matrix.After growth in the presence of a suitable culture medium for anappropriate length of time, confocal microscopy and histologicalexamination of the matrix can be conducted to determine whether thecells have grown over the surface of and/or into the matrix.

The ability of the matrix to support in-growth of nerve cells can alsobe assessed in vitro. For example, a nerve source, such as dorsal rootganglia, can be embedded into an appropriate material surrounding thematrix or directly inserted into the matrix. An example of a suitablematerial would be a soft collagen based gel. Cells from an appropriatecell line can then be seeded either directly onto the matrix or onto anappropriate material surrounding the matrix and the matrix can beincubated in the presence of a suitable culture medium for apre-determined length of time. Examination of the matrix, directlyand/or in the presence of a nerve-specific marker, for example byimmunofluorescence using a nerve-specific fluorescent marker andconfocal microscopy, for nerve growth will indicate the ability of thematrix to support neural in-growth.

Growth supplements can be added to the culture medium, to the matrix orto both in experiments to assess the ability of the matrix to supportcell growth. The particular growth supplements employed will bedependent in the type of cells being assessed and can be readilydetermined by one skilled in the art. Suitable supplements for nervecells, for example, include laminin, retinyl acetate, retinoic acid andnerve growth factors for nerve cells.

3.3 In Vivo Sting

In order to assess the biocompatibility of the bio-synthetic matrix andits ability to support cell growth in vivo, the matrix can be implantedinto an appropriate animal model for immunogenicity, inflammation,release and degradation studies, as well as determination of cellgrowth. Suitable control animals may be included in the assessment.Examples of suitable controls include, for example, unoperated animals,animals that have received allografts of similar dimensions from a donoranimal and/or animals that have received implants of similar dimensionsof a standard, accepted implant material.

At various stages post-implantation, biopsies can be taken to assesscell growth over the surface of and/or into the implant and histologicalexamination and immunohistochemistry techniques can be used to determinewhether nerve in-growth has occurred and whether inflammatory or immunecells are present at the site of the implant. For example, variouscell-specific stains known in the art can be used to assess the types ofcells present as well as various cell-specific antibodies, such aanti-neurofilament antibodies that can be used to indicate the presenceor absence of nerve cells. In addition, measurement of the nerve actionpotentials using standard techniques will provide an indication ofwhether the nerves are functional. In vivo confocal microscopicexamination can be used to monitor cell and nerve growth in the animalat selected post-operative times. Where appropriate, touch sensitivitycan be measured by techniques known in the art, for example, using anesthesiometer. Restoration of touch sensitivity indicates theregeneration of functional nerves.

4. Applications

The present invention provides a bio-synthetic matrix which is robust,biocompatible and non-cytotoxic and, therefore, suitable for use as ascaffold to allow tissue regeneration in vivo. For example, thebio-synthetic matrix can be used for implantation into a patient toreplace tissue that has been damaged or removed, for wound coverage, asa tissue sealant or adhesive, as a skin substitute or cornea substitute,or as a corneal veneer. The matrix can be moulded into an appropriateshape prior to implantation, for example it can be pre-formed to fillthe space left by damaged or removed tissue. Alternatively, when used asan implant, the matrix may be allowed to form in situ by injecting thecomponents into the damaged tissue and allowing the polymers tocross-link and gel at physiological temperature.

In one embodiment of the present invention, the matrix is pre-formedinto an appropriate shape for tissue engineering purposes. In anotherembodiment the matrix is pre-formed as a full thickness artificialcornea or as a partial thickness matrix suitable for a cornea veneer.

The bio-synthetic matrix can be used alone and as such will support thein-growth of new cells in situ. Alternatively, the matrix can be seededwith cells prior to implantation and will support the outgrowth of thesecells in vivo to repair and/or replace the surrounding tissue. It iscontemplated that the cells may be derived from the patient, or they maybe allogeneic or xenogenic in origin. For example, cells can beharvested from a patient (prior to, or during, surgery to repair thetissue) and processed under sterile conditions to provide a specificcell type such as pluripotent cells, stem cells or precursor cells.These cells can then be seeded into the matrix, as described above andthe matrix can be subsequently implanted into the patient.

The matrix can also be used to coat surgical implants to help sealtissues or to help adhere implants to tissue surfaces, for example,through the formation of cross-links between unreacted cross-linkinggroups on the synthetic polymer component of the hydrogel and primaryamino or thiol groups present in the tissue. For example, a layer of thematrix may be used to patch perforations in corneas, or be applied tocatheters or breast implants to reduce fibrosis. The matrix may also beapplied to vascular grafts or stents to minimise blood or serosal fluidleakage, to artificial patches or meshes to minimise fibrosis and tohelp adhesion of the implants to tissue surfaces.

The matrix may also be used as a delivery system to deliver a bioactiveagent to a particular region in a patient. The bioactive agent can bedelivered as a solution together with the synthetic and bio-polymerssuch that the matrix comprising the bioactive agent can form in situ, orthe matrix comprising the bioactive agent can be pre-formed andimplanted. Once within the body, the bioactive agent may be releasedfrom the matrix, for example, through diffusion-controlled processes or,if the bioactive agent is covalently bound to the matrix, by enzymaticcleavage from the matrix and subsequent release by diffusion-controlledprocesses. Alternatively, the bioactive agent may exert its effects fromwithin the matrix.

In one embodiment of the present invention, the bio-synthetic matrix isused as an artificial cornea. For this application, the matrix ispre-formed as a full thickness artificial cornea or as a partialthickness matrix suitable for a cornea veneer. In accordance with thisembodiment, the hydrogel is designed to have a high optical transmissionand low light scattering. For example, hydrogels comprising a syntheticp(NiPAAm-co-AAc-co-ASI) terpolymer or p(DMAA-co-ASI) co-polymercross-linked to collagen have high optical transmission, very low lightscattering and are capable of remaining clear up to 55° C.

5. Kits

The present invention also contemplates kits comprising thebio-synthetic matrix. The kits may comprise a “ready-made” form of thematrix or they may comprise the individual components required to makethe matrix in appropriate proportions (i.e. the synthetic polymer andthe bio-polymer. The kit may optionally further comprise one or morebioactive agent either pre-attached to the synthetic polymer, or asindividual components that can be attached to the synthetic polymerduring preparation of the matrix. The kits may further compriseinstructions for use, one or more suitable solvents, one or moreinstruments for assisting with the injection or placement of the finalmatrix composition within the body of an animal (such as a syringe,pipette, forceps, eye dropper or similar medically approved deliveryvehicle), or a combination thereof. Individual components of the kit maybe packaged in separate containers. The kit may further comprise anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of biological products, which notice reflectsapproval by the agency of the manufacture, use or sale for human oranimal applications.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It should be understood that theseexamples are for illustrative purposes only. Therefore, they should notlimit the scope of this invention in any way.

EXAMPLES

Abbreviations

-   RTT: rat-tail tendon-   ddH₂O: distilled, de-ionised water-   PBS: phosphate buffered saline-   D-PBS: Dulbecco's phosphate buffered saline-   AIBN: azobis-isobutyronitrile-   NiPAAm: N-isopropylacrylamide-   pNiPAAm: poly(N-iso-propylacrylamide)-   AAc: acrylic acid-   DMAA N,N-dimethylacrylamide-   ASI: N-acryloxysuccinimide-   pNIPAAm-co-AAc: copolymer of NiPAAm and AAc-   poly(NiPAAm-co-AAc-co-ASI): terpolymer of NIPAAM, AAc and ASI-   poly(DMAA-co-ASI) co-polymer of DMAA and ASI-   GPC: gel permeation chromatography-   NMR nuclear magnetic resonance-   YIGSR: amide-terminated pentapeptide    (tyrosine-isoleucine-glycine-serine-arginine)

All gel matrices described in the Examples set out below used sterilecollagen I, such as telocollagen (rat-tail tendon, RTT) or atelocollagen(bovine or porcine), which can be prepared in the laboratory or moreconveniently is available commercially (for example, from BectonDickinson at a concentration of 3.0-3.5 mg/ml in 0.02N acetic acid andin 0.012N hydrochloric acid for bovine and porcine collagen). Suchcollagens can be stored for many months at 4° C. In addition, suchcollagen solutions may be carefully concentrated to give opticallyclear, very viscous solutions of 3-30 wt/vol % collagen, suitable forpreparing more robust matrices.

Collagen solutions are adjusted to physiological conditions, i.e. salineionic strength and pH 7.2-7.4, through the use of aqueous sodiumhydroxide in the presence of phosphate buffered saline (PBS). PBS, whichis free of amino acids and other nutrients, was used to avoid depletionof cross-linking reactivity by side reactions with —NH₂ containingmolecules, so allowing the use of the minimum concentration ofcross-linking groups and minimising any risk of cell toxicity.

pNiPAAm homopolymer powder is available commercially (for example, fromPolyscience). All other polymers were synthesized as outlined below.

Example 1 Preparation of a pNiPAAm-Collagen Hydrogel

A pNiPAAm-collagen hydrogel was prepared to provide an alternativehydrogel against which the properties of the hydrogels of the presentinvention could be compared.

A 1 wt/vol % solution of pNiPAAm homopolymer in ddH₂O was sterilised byautoclaving. This solution was mixed with sterile RTT collagen solution[3.0-3.5 mg/ml (w/v) in acetic acid (0.02N in water] (1:1 vol/vol) in asterile test tube at 4° C. by syringe pumping to give complete mixingwithout bubble formation. Cold mixing avoids any premature gelificationor fibrilogenesis of the collagen. The collagen-pNiPAAm was then pouredover a plastic dish (untreated culture dish) or a mould (e.g. contactlens mould) and left to air-dry under sterile conditions in a laminarflow hood for at least 2-3 days at room temperature. After drying toconstant weight (˜7% water residue), the formed matrix was removed fromthe mould. Removal of the matrix from the mould is facilitated bysoaking the mould in a sterile PBS at room temperature. Continuedsoaking of the free sample in this solution gives a gel at physiologicaltemperature, pH and ionic strength, which was subsequently submitted totesting for cell growth and in vivo animal testing (see Examples 6 and7).

Example 2 Preparation of a Synthetic Terpolymer

A collagen-reactive terpolymer, poly(NiPAAm-co-AAc-co-ASD (FIG. 1), wassynthesised by co-polymerising the three monomers:N-isopropylacrylamide, (NiPAAm, 0.85 mole), acrylic acid (AAc, 0.10mole) and N-acryloxysuccinimide (ASI, 0.05 mole). The feed molar ratiowas 85:10:5 (NiPAAm: AAc: ASI), the free-radical initiator AIBN (0.007mole/mole of total monomers) and the solvent, dioxane (100 ml), nitrogenpurged before adding AIBN. The reaction proceeded for 24 h at 65° C.

After purification by repeated precipitation to remove traces ofhomopolymer, the composition of the synthesised terpolymer (82% yield)was found to be 84.2:9.8:6.0 (molar ratio) by proton NMR in THF-DG. TheM_(n) and M_(w) of the terpolymer were 5.6×10⁴ Da and 9.0×10⁴ Da,respectively, by aqueous GPC.

A solution of 2 mg/ml of the terpolymer in D-PBS remained clear even upto 55° C., consistent with a high LCST. A solution of 10 mg/ml in D-PBSbecame only slightly cloudy at 43° C. Failure to remove homopolymerformed in the batch polymerisation reaction (due to the NiPAAmreactivity coefficient being greater than that of AAc or ASI) from theterpolymer gave aqueous solutions and hydrogels which cloud at ˜32° C.and above.

Example 3 Preparation of a Synthetic Polymer Comprising a BioactiveAgent

A terpolymer, containing the pentapeptide YIGSR (a nerve cell attachmentmotif), was synthesised by mixing the terpolymer prepared in Example 2(1.0 g) with 2.8 μg of laminin pentapeptide (YIGSR, from Novabiochem) inN,N-dimethyl formamide. After reaction for 48 h at room temperature (21°C.), the polymer product was precipitated out from diethyl ether andthen vacuum dried. ASI groups remaining after reaction with thepentapeptide are available for subsequent reaction with collagen. Thestructure of this polymer is shown in FIG. 8A.

Example 4 Preparation of a Collagen-Terpolymer Hydrogel

A cross-linked, terpolymer-collagen hydrogel was made by mixingneutralised 4% bovine atelocollagen (1.2 ml) with the terpolymerprepared in Example 2 [0.34 ml (100 mg/ml in D-PBS)] by syringe mixingat 4° C. (collagen:terpolymer 1.4:1 w/w). After careful syringe pumpingto produce a homogeneous, bubble-free solution, aliquots were injectedinto plastic, contact lens moulds and incubated at room temperature (21°C.) for 24 hours to allow reaction of the collagen —NH₂ groups with ASIgroups as well as the slower hydrolysis of residual ASI groups to AAcgroups. The moulded samples were then incubated at 37° C. for 24 hoursin 100% humidity environment, to give a final hydrogel. The hydrogelcontained 95.4±0.1% water, 2.3% collagen and 1.6% terpolymer. Matriceswere moulded to have a final thickness between either 150-200 μm or500-600 μm. Each resulting hydrogel matrix was removed from its mouldunder PBS solution and subsequently immersed in PBS containing 1%chloroform and 0.5% glycine. This wash step removed N-hydroxysuccinimideproduced in the cross-linking reaction, terminated any unreacted ASIgroups in the matrix, by conversion to acrylic acid groups andsterilised the hydrogel matrix. As an alternative, moulded gels may betreated with aqueous glycine to ensure that all ASI are terminated priorto cell contact.

Succinimide residues left in the gels prepared from collagen andterpolymer were below the R detection limit after washing.

Example 5 Preparation of a Hydrogel Comprising a Bioactive Agent

Cross-linked hydrogels of collagen-terpolymer comprising YIGSR celladhesion factor were prepared by thoroughly mixing viscous, neutralised4% bovine collagen (1.2 ml) with terpolymer to which lamininpentapeptide (YIGSR) was covalently attached (from Example 3; 0.34 ml,100 mg/ml) at 4° C., following the procedure described in Example 4.

The YIGSR content of extensively washed gels was 4.3×10⁻¹¹ mole/ml(2.6×10⁻⁸ g/ml) of hydrated gel quantified by labelling the tyrosine(primary amine-bearing) groups with ¹²⁵I using the Iodogen method andmeasuring the radioactivity with a standardised gamma counter (Beckman,Gamma 5500). The final, total polymer concentration in each hydrated,PBS-equilibrated hydrogel was 3.4 w/v % (comprising collagen and YIGSRterpolymer at 2.0 and 1.4 w/v %, respectively).

Example 6 Comparision of the Physical Properties of Hydrogel Matrices

Collagen thermogels are frail and readily collapse and break and areobviously opaque (see FIG. 8C). Collagen thermogels were prepared byneutralization of collagen and casting in the same moulds as describedabove in Examples 4 and 5. The moulded collagen was then incubated,first for 24 h at 21° C. then at 37° C., to spontaneously formtranslucent thermogels (produced by self association of collagen triplehelices into micro-fibrils).

The permeability coefficient of glucose in PBS (pH 7.4) throughhydrogels prepared as described in Examples 5 was calculated frommeasurements in a permeation cell by periodically removing aliquots ofpermeate, adding adenosine triphosphate and converting glucose toglucose-6-phosphate with the enzyme hexokinase. The latter was reactedwith nicotinamide adenine dinucleotide in the presence of dehydrogenaseand the resultant reduced dinucleotide quantified by its UV absorptionat 340 nm in solution (Bondar, R. J. & Mead, D. C. (1974) Clin Chem 20,586-90). Topographies of hydrogel surfaces, fully immersed in PBSsolution, were examined by atomic force microscopy (Molecular Image Co.,USA) in the “contact” mode. Pore sizes from this technique were comparedwith average pore diameters calculated from the PBS permeability of thehydrogels as previously described (Bellamkonda, R., Ranieri, J. P. &Aebischer, P. (1995) J Neurosci Res 41, 501-9). The hydrogels hadrefractive indices (1.343±0.003) comparable to the tear film(1.336-1.357) in the human eye (Patel, S., Marshall, J. & Fitzke, F. W.,3rd (1995) J Refract Surg 11, 100-5). They showed high optical claritycompared to matrices that contain only collagen (FIGS. 8B and C). Thehydrogels had pore diameters of 140-190 nm (from both atomic forcemicroscopy and PBS permeability) and a glucose diffusion permeabilitycoefficient of 2.7×10⁻⁶ cm²/s, which is higher than the value for thenatural stroma (˜0.7×10⁻⁶ cm²/s, calculated from published diffusion(2.4×10⁻⁶ cm²/s) and solubility (0.3) coefficients (McCarey, B. E. &Schmidt, F. H. (1990) Curr Eye Res 9, 1025-39)).

The following properties of the hydrogels prepared as described inExamples 4 and 5 indicate that they are cross-linked:

-   -   water insoluble,    -   strong enough to support surgical manipulation with suture        thread and needle, and attachment to a human corneal ring    -   relatively flexible in handling    -   demonstrate an increase in stress at failure and apparent        modulus during tensile testing by over ×2 on going from ASI/-NH₂        equivalent ratio of 0.5 to 1.5.

Matrices prepared as described above, but with varying ratios ofcollagen amine groups to ASI groups in the synthetic polymer had highoptical transmission and low scatter in the visible region (FIGS. 8B, 12and 13). In contrast, the collagen thermogel, prepared from collagen asdescribed above, had low transmission and high scatter, consistent withits opaque appearance (FIGS. 8C, 12). Such thermogel matrices with up to3 wt/vol collagen were too weak to allow mounting for suture pull-outtesting. Quantitative characterisation of the hydrogels came from theuse of suture pull-out measurements on samples moulded into the shapeand thickness of a human cornea. This involved insertion of twodiametrically opposed sutures, as required for the first step in ocularimplantation, and pulling these two sutures apart at 10 mm/min on anInstron Tensile Tester, a procedure that is well established for theevaluation of heart valve components. The sutures employed were 10-0nylon sutures. Strength at rupture of the gel is calculated, togetherwith elongation at break and elastic modulus. Modulus and stress atfailure from suture pull-out measurments showed that maxima were reachedat specific collagen amine to ASI group ratios (FIG. 11).

The hydrogels prepared as described in Examples 4 and 5 have highoptical transmission and very low light scattering, comparable to thehuman cornea, as measured with a custom-built instrument that measurestransmission and scatter [Priest and Munger, Invest. Ophthalmol. Vis.Sci. 39: S352- S361 (1998)]. Back scattering and transmission of lightacross the visible region for hydrogels prepared as in Example 4 showedexcellent performance except at high terpolymer concentrations (highcollagen amine to terpolymer ASI ratios, FIG. 12). Similarly, athermogel (free of cross-linking synthetic polymer) had a very lowtransmission and high back scattering (FIG. 12). The hydrogels describedin Example 5 also showed excellent performance in this analysis as shownin FIG. 12 (1:1 ratio of collagen to terpolymer-pentapeptide isrepresented by the solid squares).

In contrast, collagen-pNiPAAm homopolymer gels (as described in Example1; 1.0:0.7 to 1.0:2.0 wt/wt) were opaque at 37° C. In addition, bothcollagen and pNiPAAm extracted out from this hydrogel into PBS (over 50wt % loss in 48 h).

Example 7 In Vivo Testing of Various Bio-Synthetic Matrices

Hydrogels formed as described in Examples 1, 4 and 5 were moulded toform artificial corneas and implanted into the eyes of pigs (FIG. 2).

As in vivo corneal implants, the gels from Example 1 exude white residueafter 5 to 6 days implanted in pigs' eyes.

The hydrogel prepared from 4% collagen and pentapeptide-terpolymer asdescribed in Example 5 demonstrated good biocompatibility as did thecollagen-terpolymer hydrogel prepared as described in Example 4. Morerapid, complete epithelial cell overgrowth and formation of multiplelayers occurred when the former hydrogel was used, as compared tocollagen-terpolymer hydrogel which showed slower, less contiguous,epithelial cell growth, without formation of multiple layers.

In vivo, confocal microscope images of full thickness hydrogel preparedfrom collagen and the pentapeptide-terpolymer (from Example 5; finalconcentration: collagen 2.3 wt %; terpolymer+pentapeptide 1.6 wt %) andimplanted into a pig's eye showed that epithelium cells grew over thismatrix and stratified. A basement membrane was regenerated andhemidesmosomes, indicating a stably anchored epithelium, were present.Stromal cells were found to spread inside the matrix after only threeweeks. The implants became touch sensitive within 3 weeks ofimplantation (Cochet-Bonnet Aesthesiometer) indicating functional nervein-growth (FIGS. 4 and 14). Nerve in-growth was also observed directlyby confocal microscopy and histology. No clinical signs of adverseinflammation or immune reaction were observed over an 8 week periodfollowing implantation. See FIGS. 2, 3 and 5-7.

In more detail:

FIG. 5 shows morphological and biochemical assessment of a sectionthrough the pig cornea at 3 weeks post-implantation (A) picro-siriusstain for collagen and (B) H&E stain for cells. FIG. 7 shows (A) asection through the pig cornea at 3 weeks post-implantation, stainedwith picro-sirius red, which demonstrates the stromal-implant interface(arrowheads). The implant surface has been re-covered by a stratifiedepithelium. (B) a similar section at 8 weeks post-implantation. Stromalcells have moved into the implant and the implant appears to have beenreplaced by tissue sub-epithelially (arrows). (C) a higher magnificationof the epithelium (H & E stained) showing the regenerated basementmembrane (arrow). (D) a corresponding section stained with anti-type VIIcollagen antibody that recognizes hemidesmosomes attached to thebasement membrane (arrow). (E) the hemidesmosomes (arrows) attached tothe underlying basement membranes are clearly visualized by transmissionelectron microscopy (TEM). (F) a flat mount of the pig cornea showingnerves (arrowheads) within the implant, stained with ananti-neurofilament antibody. FIG. 3 shows whole mount confocalmicroscopic images of pigs corneas at 6 weeks post-operation showing aregenerated corneal epithelium and basement membrane on the surface ofthe implant. In vitro nerve growth patterns within thecollagen-terpolymer composite and within the underlying host stroma areshown, as are in-growing stromal cells.

Restoration of touch sensitivity was rapid (<14 days post-operative) incomparison with minimal restoration in the transplanted allograft overthe same time period for an additional six animals that receivedallografts of donor pig corneas of similar dimensions (FIG. 14).

Example 8 Deposition of Collagen-Terpolymer Matrices into Rodent Brain

Following euthanasia, the whole brain of each mouse or rat used wasexcised and placed within a sterotaxic frame. Either two microlitres (2ml) or three microlitres (3 ml) of hydrogel containing collagen,terpolymer-pentapeptide at either 0.33% collagen −0.23% terpolymer or0.63% collagen−0.44% terpolymer was injected over a period of 6 to 10min, respectively, into each individual mouse brain, at the followingcoordinates: 0.3 mm from bregma, 3.0 mm deep and 2.0 mm from themidline. For rats, four to six microlitres of hydrogel was injected over10 min. into each brain, at 0.7-0.8 nm from bregma, 6 mm deep and 4 mmfrom the midline. The hydrogel samples were mixed with Coomassie bluedye for visualization.

Results indicate successful direct, precise delivery of small amount ofthe hydrogel into the stratum of the brain, in these samples (FIGS. 9and 10). This suggests that it is possible to use the hydrogel as adelivery system for cells or drugs into specific locations at very smallvolumes.

Example 9 In Vivo Testing of a Hydrogel Comprising a Bioactive Agent

Sterile hydrogels prepared as described in Example 5 were thoroughlyrinsed in PBS before implantation. Following the Association forResearch in Vision and Ophthalmology guidelines for animal use, eachtissue engineered (TE) corneal matrix (5.5 mm in diameter and 200±50 μmthick) was implanted into the right cornea of a Yucatan micro-pig(Charles River Wiga, Sulzbach, Germany) (see FIGS. 15A-C). Contralateralunoperated corneas served as controls. Under general anaesthesia, apartial-thickness 5.0 mm diameter circular incision was made using aBarraquer trephine (Geuder, Heidelberg, Germany). Host corneal tissuewas removed and replaced with an implant 0.50 mm larger in diameter toallow adequate wound apposition between the graft and host tissue. Aftersurgery, an amniotic membrane was sutured over the entire cornealsurface for one week to keep implants in place. In sutured samples,implants were sutured into the host tissue using 8 interrupted 10-0nylon sutures. Post-operative medication consisted of dexamethasone(qid) and gentamycin (qid) for 21 days. n=3 pigs with sutures and 3without sutures.

Follow-ups were performed daily on each pig up to 7 days post-operative,and then weekly. Examinations included slit-lamp examination to ensurecorneas were optically clear, sodium fluorescein staining to assessepithelial integrity and barrier function (Josephson, J. E. & Caffery,B. E. (1988) Invest Ophthalmol Vis Sci 29, 1096-9), measurements ofintraocular pressure to ensure that corneas were not blocking aqueoushumour flow, and in vivo confocal microscopic examination (ConfoScan3,Nidek, Erlangen, Germany) to assess cell and nerve in-growth. Cornealtouch sensitivity was measured using a Cochet-Bonnet esthesiometer(Handaya Co., Tokyo, Japan) at five points within the implant area ofeach cornea (four peripheral, one central) as previously described(Millodot, M. (1984) Ophthalmic Physiol Opt 4, 305-18). Animals thatreceived allografts of pig donor corneas were also similarly evaluated.

For immunohistochemistry and histopathological examination, tissues andconstructs 20 were fixed in 4% PFA in 0.1M PBS. For nerveimmunolocalization, flat mounts were permeabilized with a detergentcocktail (Brugge, J. S. & Erikson, R. L. (1977) Nature 269, 346-8) (150mM NaCl, 1 mM ethylenediamine tetraacetic acid, 50 mM Tris, 1% NonidetP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate), blockedfor non-specific staining with 4% foetal calf serum in PBS and incubatedin anti-neurofilament 200 antibody (Sigma, Oakville, Canada). They werethen incubated with FITC or Cy3-conjugated secondary antibodies (Sigma;Amersham, Baie D'Urfé, Canada, respectively) and visualization byconfocal microscopy.

For histology and further immunohistochemistry, samples were processed,paraffin embedded and sectioned. Sections were stained with haematoxylinand eosin (H&E) for histopathological examination (Jumblatt, M. M. &Neufeld, A. H. (1983) Invest Ophthalniol Vis Sci 24, 1139-43).Immunofluorescence was performed as described above on deparaffinizedsections for expression of type VII collagen (Sigma, Munich, Germany), ahemidesmosome marker (Gipson, I. K., Spurr-Michaud, S. J. & Tisdale, A.S. (1988) Dev Biol 126, 253-62). Immunohistochemical staining usingperoxidase-diaminobenzidine (DAB) visualization was performed with thefollowing: with AE1/AE3 antibody (Chemicon, Temecula, Calif., USA) forepithelial markers, anti-vimentin antibody (Roche, Laval, Canada) forstromal fibroblasts, anti-smooth muscle actin antibody, 1A4 (CellMarque, Austin, Tex.) for activated stromal fibroblasts (myofibroblasts)and SP1-D8 antibody (DHSB, Iowa, USA) for procollagen 1 synthesis (tolocalize sites of de novo collagen synthesis). CD15 and CD45 stainingfor immune cells (Becton-Dickinson, Oakville, Canada) was performedusing the ARK peroxidase kit (DAKO, Mississauga, Canada) topre-conjugate the primary antibodies to their respective secondaryantibodies and peroxidase for visualization. For anti-vimentin,anti-smooth muscle actin and SP8-D1 antibodies, antigen retrieval waspreformed by pre-treating with Proteinase-K (2 mg/ml) for 30 min at 37°C. prior to incubation in primary antibody. Ulex europaeus aggultinin(UEA) lectin staining was used to visualize tear film mucin deposition(Shatos, M. A., Rios, J. D., Tepavcevic, V., Kano, H., Hodges, R. &Dartt, D. A. (2001) Invest Ophthalmol Vis Sci 42, 1455-64). Samples wereincubated with biotinylated UEA (Sigma), then reacted withavidin-horseradish peroxidase and visualized with DAB. For transmissionelectron microscopy (TEM), all samples were treated in conventionalfixative, stain and potting resin (Karnovsky's, OsO₄, uranyl acetate,epoxy).

No adverse inflammatory or immune reaction was observed by clinicalexamination after implantation of either bio-synthetic matrices or pigcorneas. Epithelial cell in-growth over the implant was complete by 4days post-operative. By one week, the regenerated epithelium showedexclusion of sodium fluorescein dye, indicating that the epithelium wasintact and had re-established barrier properties. Intraocular pressureswere between 10 and 14 mm mercury (Hg) pre-operatively, and 10-16 mm Hgpost-operatively throughout the study period of up to 6 weeks, showingthat the implants did not block flow of aqueous humour within the eye.Implants remained optically clear (slit-lamp biomicroscopy) andepithelial re-stratification was observed in all animals at 3 weekspost-surgery. Clinical in vivo confocal microscopy of the implantedstromal matrices at 3 weeks post-surgery showed a regenerated epithelium(FIG. 15D), newly in-grown nerves (FIG. 15G), and stromal (FIG. 15J) andendothelial cells (FIG. 15M) with cellular morphology mimicking that ofun-operated controls (FIGS. 15F,I,L,O). Epithelial and endothelial cellmorphology in the allografts (FIGS. 15E,N) was similar to that ofcontrols. Sub-epithelial and stromal nerves were not observed in theallografts at 3 weeks post-surgery (FIGS. 15H,K).

In more detail, FIG. 15 shows:

(A-C) lamellar keratoplasty (LKP) procedure on a Yucatan micro-pig. (A):A trephine is used to cut a circular incision of pre-set depth (250 μm)into the cornea. The existing corneal layers are removed and (B) arereplaced with a bio-synthetic matrix implant (arrow, 250 μm inthickness), which is sutured in place (C). Sutures are indicated byarrowheads.

(D-O) In vivo confocal microscopy of implanted bio-synthetic matrix.(D): confocal image showing regenerated corneal epithelium on thesurface of the implant. The corresponding allograft control (E) containsdonor epithelium, while the un-operated control (F) has an intactepithelium. (G): Regenerated nerves (arrowheads) are present at theinterface between implant and overlying regenerated epithelium. Thesecorrespond to the sub-epithelial nerves in the un-operated control (I).In the allograft (H), however, sub-epithelial nerves are absent. (J-L):Stromal cells and branching nerve bundle (arrowhead) deeper within theunderlying stroma of corneas with implant (B), allograft (K) and in acorresponding region in the control (L). (M-O): The endothelium incorneas with implant (M), allograft (N) and un-operated controls (O) areintact and show similar morphology.

Histological sections through corneas with implants showed a distinctbut smooth, implant-host tissue interface (FIG. 16A) that resembled thatof control corneas that received allografts (FIG. 16B). In both corneaswith implants or allografts, the regenerated epithelium was stratified.Detailed examination showed a fully differentiated epithelium that waspositively stained by AE1/AE3 antibody markers (FIGS. 16D,E), overlyinga regenerated basement membrane that was positive for Type VII collagen,a marker for hemidesmosomes at the basement membrane-epitheliuminterface (FIGS. 16G,H). TEM observations indicated morphologyconsistent with the presence of hemidesmosomes (FIGS. 16J,K). In theimplants, neurofilament-positive in-growing nerves had begun tore-establish a sub-epithelial network and showed extension into theepithelial cells (FIG. 16M). However, no sub-epithelial nerves werelocated in the allografted corneas (FIG. 16N). The tear film wasrestored in corneas with implants (FIG. 16P) as in the allograft (FIG.16Q).

In more detail, FIG. 16 shows: post-surgical corneal regeneration.

(A-F) H&E stained sections are shown. Stromal cells are present in theimplant (A) and the allograft control (B), and both appear to beseamlessly integrated into the host. (symbols are as follows: e,epithelium; i, implant; g, allograft; s, stroma). (C): Unoperatedcontrol. The regenerated epithelium of the implant (D) and donorepithelium of the allograft control (E) expressed cytokeratindifferentiation markers, similar to the un-operated control (F).

(G-I): Immunolocalization of type VII collagen, a marker forhemidesmosomes, at the epithelium-implant interface (arrows) in theimplant (G), allograft (H) and control (I).

(J-L): TEM of epithelium-implant interface. Hemidesmosome plaques(arrowheads) and anchoring fibrils (arrows) have formed within thebio-synthetic matrix between the epithelial cells and underlying implant(J), emulating the structures normally found at the epithelial-stromalinterface as demonstrated in the allograft (K) and control (L).

Flat mount of cornea showing nerve fibres (arrows) within the implant(M), and un-operated control (O) but absent in the allograft (N),stained with an anti-neurofilament antibody. UEA binding (arrowheads) tothe epithelial surface on the implant (P), and allograft (Q) indicaterestoration of the tear film in all cases. Un-operated control (R).

Immunohistochemistry results indicated that cells within both implantand allograft were synthesizing procollagen I. However, more procollagensynthesis occurred in the allografts as indicated by the more intensestaining in allografts compared to implants (FIGS. 17G,H). Bothallografts and implants had stromal cells that were vimentin positive(FIGS. 17A,B), indicating a fibroblastic phenotype. Both also showedsmooth muscle actin staining and therefore the presence of activatedstromal fibroblasts, although the implants showed fewer positive cellsthan the allografts (FIGS. 17D,E).

In more detail, FIG. 17 shows: implant-host integration at 6 weeks postsurgery.

(A-C): Staining for procollagen type I. Positive staining is observed inmatrix of both the implanted biosynthetic matrix (A) and the allograftcontrol (B) indicating sites of new collagen deposition. Unoperatedcontrol (C) has no new collagen synthesis.

(D-F): Staining for vimentin throughout stroma identifies stromalfibroblasts. Staining throughout the implanted biosynthetic matrix (D)demonstrates cell invasion. Cells may also been seen within theimplanted allograft (E) and throughout the un-operated control (F).

(G-I): Smooth muscle actin staining indicates activated myofibroblastsand the potential for scarring. In the biosynthetic matrix implant (G),staining is occasionally present in the biosynthetic matrix, but is notfound in the host stroma, nor in the transition zone between host andimplant. Positive staining in the allograft implanted cornea (H) isidentified both in the allograft, and the transition zone, but not inthe intact host stroma. (I): Un-operated control.

Corneal touch sensitivity measured at 5 points on the corneal implant in3 pigs pre- and post-operatively using a Cochet-Bonnet esthesiometer,showed a dramatic drop in touch sensitivity after surgery. However,recovery occurred between 7 and 14 days and by 21 days post-operative,sensitivity had returned to pre-operative levels (FIG. 18; All groups,n=3. *P<0.01 by repeated measures ANOVA with Tukey 2-way comparisons).Touch sensitivity returned at the same rate and to the same plateaulevel at all peripheral and central points tested on the implant. Incontrol animals that had received donor corneal allografts, however, thecorneas remained anaesthetic over the six-week period (FIG. 18).

Implants recovered after 6 weeks in vivo were examined by infraredspectroscopy Midac M, FTIR spectrometer, ZnSe beam condenser and diamondcell) and clearly indicated the presence of the terpolymer.

Example 10 Preparation of a Synthetic Co-Polymer

A poly(DMAA-co-ASI) co-polymer was synthesised by co-polymerization ofthe monomers: N,N-dimethyl acrylamide, (DMAA) and N-acryloxysuccinimide(ASI). The feed molar ratio was 95:5 (DMAA: ASI). The free-radicalinitiator AIBN and the solvent, dioxane, were nitrogen purged prior touse and polymerisation reaction proceeded at 70° C. for 24 h.

After purification by repeated precipitation to remove traces ofhomopolymer, the composition of the synthesized copolymer (70% yield)was found to be 94.8:5.2 (molar ratio) by proton NMR. Molecular mass(M_(n)) was determined at 4.3×10⁴, by aqueous GPC. Polydispersity (PD;M_(w)/M_(n))=1.70 was also determined by GPC.

Example 11 Preparation of a Collagen-Co-Polymer Hydrogel

A cross-linked collagen-co-polymer hydrogel was prepared by mixingneutralized 5% bovine collagen (1.0 ml) with the synthetic co-polymerprepared in Example 9 [0.2 ml (200 mg/ml in D-PBS)] by syringe mixing.After careful syringe pumping to produce a homogeneous, bubble-freesolution, aliquots were injected into plastic, contact lens moulds andincubated at room temperature for 24 hours to allow reaction of thecollagen —NH₂ groups with ASI groups in the co-polymer as well as theslower hydrolysis of residual ASI groups to AAc groups.

The moulded samples were then incubated at 37° C. for 24 hours in a 100%humidity environment to provide the final hydrogel. At gelation, thehydrogel contained 94.8% water, 2.9% collagen and 2.3% syntheticco-polymer. Matrices were moulded to have a final thickness betweeneither 150-200 μm or 500-600 μm. Each resulting hydrogel matrix wasremoved from its mould under PBS solution and subsequently immersed inPBS containing 1% chloroform and 0.5% glycine. This wash step removedN-hydroxysuccinimide produced in the cross-linking reaction andterminated any residual ASI groups in the matrix, by conversion toacrylic acid groups.

Succinimide residues left in the gels prepared from collagen andcopolymer were below the IR detection limit after washing.

Example 12 Physical Properties of Collagen-Co-Polymer Hydrogel

Light back scattering and light transmission across the visible regionand with white light for hydrogels prepared as in Example 10 as afunction of collagen amine to copolymer ASI ratios is shown in FIGS. 13Aand B.

The copolymer from Example 10 and its hydrogels had no detectable cloudpoint (LCST) at up to 60° C.

Example 13 Biological Properties of Various Hydrogels

11.1 Biocompatibility

Three 12 mm diameter and 650 μm thick discs each ofcollagen-poly(DMAA-co-ASI),collagen-poly(NiPAAm-co-AAc-co-ASD-pentapeptide and 3% collagenhydrogels were soaked for 30 minutes in PBS. They were each laid onto a12 mm membrane insert commercially available for a culture dish andadhered to the membrane with a thin coating of gelatin. After drying for10 minutes, 1×10⁴ human corneal epithelial cells (HCEC) cells suspendeda serum-free medium containing epidermal growth factor (KeratinocyteSerum-Free Medium (KSFM; Life Technologies, Burlington, Canada)) wereadded to the top of the gels, and KSFM without cells was added to theunderlying well. Cultures were incubated at 37° C. with 5% CO₂.

Within 12 hours the cells had adhered to the surface of the matrix inall samples. Medium was changed every second day with KSFM added to theinserts and to the outside wells. HCEC were grown to confluence on thegels and reached confluence on the same day (5 days). The medium in theinserts and surrounding wells was replaced by a serum-containing medium(modified SHEM medium (Jumblatt, M. M. & Neufeld, A. H. (1983) InvestOphthalinol Vis Sci 24, 1139-43)). After 2 more days, the medium wasremoved from the inserts, and the volume of SHEM in the underlying wellsreduced to 0.5 ml. The epithelium was allowed to stratify for a further7 days and the layer of cells visualized.

After 7 days, the membranes were fixed in 4% paraformaldehyde in PBS for30 minutes at 4° C. Samples were prepared for cryosectioning byequilibration in 30% sucrose in PBS followed by flash freezing in a 1:1mixture of 30% sucrose in PBS and OCT. These were cryosectioned to 13 μmand the structure visualized by HandE staining. The number of celllayers in the stratified epithelium was determined by counting nucleiand identifying cell borders. The collagen thermogel attained anepithelial thickness of approximately 2 cells, which contrasts poorlywith the human corneal epithelium that contains between about 5 and 7cell layers. HCEC cultured and induced to stratify oncollagen-p(DMAA-co-ASI) andcollagen-p(NiPAAm-co-AAc-co-ASI)-pentapeptide, however, resulted in anepithelium about 4.5 cell layers thick that included apparentlykeratinized outer layers suggesting appropriate differentiation of theepithelium (FIG. 20).

11.2. Innervation of Hydrogel

Twelve millimeter diameter and 650 μm thick discs each ofcollagen-p(DMAA-co-ASI), collagen-(NiPAAm-co-AAc-co-ASI)-pentapeptideand 3% collagen thermogel were soaked for 30 minutes in PBS. Discs werelaid in a 6 cm culture dish, and four 1 mm holes bored through each. Theholes were filled a third of the way up with a plug of 0.3% collagencross-linked with glutaraldehyde and quenched with glycine. After 10minutes, dorsal root ganglions from E8 chicks were dipped in the samecollagen mixture and placed in the holes. The holes were filled the restof the way with cross-linked collagen, and allowed to set for 30 minutesat 37° C. Cultures were grown for 4 days in KSFM supplemented with B27,N2, and 1 nM retinoic acid for 4 days and neurite extension monitored bybrightfield microscopy. The innervated discs were fixed in 4%paraformaldehyde in PBS for 30 minutes room temperature, stained forNF200 immunoreactivity, and visualized by immunofluorescence.Localization was visualized on the surface and in the centre of thepolymer disc. While there was some neurite extension over the surface ofthe collagen thermogel, none could be seen extending into the thermogelitself. In the hydrogels, neurites could be seen extending into thematrix. As well, in both the hydrogels extensive innervation could beseen over the surface of the matrix suggesting a better surfaceinnervation than occurred with the collagen thermogel (FIG. 19; Adepicts the collagen thermogel, B depicts thecollagen-p(iPAAm-co-AAc-co-ASI)-pentapeptide hydrogel and C depicts thecollagen-p(DMAA-co-ASI) hydrogel). The left column representsimmunofluorescent visualizations of the middle of the polymers stainedfor the nerve neurofilament marker—NF200. The middle column depicts abrightfield view of the surface of the polymer with the neuritesextending from the ganglion source. The right column represents animmunofluorescent visualization of the same surface view of the polymerstained for NF200 immuno-reactivity. The arrows indicate neuritesextending in the middle of the polymer. The intact human corneademonstrates both sub-epithelial surface and deep nerves suggesting thatthese matrices are both biocompatible to nerves and can emulate thecorneal stroma.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. A synthetic co-polymer comprising one or more N-alkyl or N,N-dialkylsubstituted acrylamide co-monomer, one or more hydrophilic co-monomerand one or more acryl- or methacryl-carbokylic acid co-monomerderivatised to contain a pendant cross-linkable moiety, said syntheticco-polymer having a number average molecular mass between about 2,000and about 1,000,000, wherein said synthetic co-polymer is reactive withprimary amines via the pendant cross-linkable moiety.
 2. The syntheticco-polymer according to claim 1, wherein: (a) said N-alkyl orN,N-dialkyl substituted acrylamide co-monomer has a structure of FormulaI:

wherein: R₁, R₂, R₃, R₄ and R₅ are independently selected from the groupof: H and lower alkyl; (b) said hydrophilic co-monomer has a structureof Formula II:

wherein: Y is O or is absent; R₆, and R₇ are independently selected fromthe group of H and lower alkyl; R₈ is H, lower alkyl or —OR′, where R′is H or lower alkyl; and R₉ is H, lower alkyl or —C(O)R₁₀, and R₁₀ is—NR₄R₅ or —OR″, where R″ is H or CH₂CH₂OH; and (c) said acryl- ormethacryl-carboxylic acid co-monomer has a structure of Formula III:

wherein: R₁₁, R₁₂ and R₁₃ are independently selected from the group of:H and lower alkyl, and Q is N-succinimido, 3-sulpho-succinimdo (sodiumsalt), N-benzotriazolyl, N-imidazolyl and p-nitrophenyl.
 3. Thesynthetic co-polymer according to claim 1, wherein said one or moreN-alkyl or N,N-dialkyl substituted acrylamide co-monomer and said one ormore hydrophilic co-monomer are the same.
 4. The synthetic co-polymeraccording to claim 1, wherein said one or more N-alkyl or N,N-dialkylsubstituted acrylamide co-monomer and said one or more hydrophilicco-monomer are different.
 5. The synthetic co-polymer according to claim1, wherein said alkyl or lower alkyl is a straight or branched chainalkyl group having between one and eight carbon atoms.
 6. The syntheticco-polymer according to claim 2, wherein the combined molar ratio ofN-alkyl or N,N-dialkyl substituted acrylamide co-monomer and hydrophilicco-monomer is between about 50% and about 99.5% and the molar ratio ofderivatised acryl- or methacryl-carboxylic acid co-monomer is betweenabout 0.5% and about 50%, wherein the sum of said molar ratios is 100%.7. The synthetic co-polymer according to claim 3, wherein the molarratio of N-alkyl or N,N-dialkyl substituted acrylamide co-monomer isbetween about 50% and about 90%, the molar ratio of the hydrophilicco-monomer is between about 5% and about 50% and the molar ratio ofderivatised acryl- or methacryl- carboxylic acid co-monomer is betweenabout 0.1% and about 15%, wherein the sum of said molar ratios is 100%.8. The synthetic co-polymer according to claim 1, wherein said one ormore N-alkyl or N,N-dialkyl substituted acrylamide co-monomer isselected from the group of N-methylacrylamide, N-ethylacrylamide, Nisopropylacrylamide (NiPAAm), N-octylacrylamide, N-cyclohexylacrylamide,N-methyl-N-ethylacrylamide, N-methylmethacrylamide,N-ethylmethacrylamide, N-isopropylmethacrylamide,N,N-dimethylacrylamide, N,N-diethylacrylamide,N,N-dimethylmethacrylamide, N,N-diethylmethacrylamide,diethylmethacrylamide, N,N-dicyclohexylacrylamide,N-methyl-N-cyclohexylacrylamide, N-acryloylpyrrolidine,N-vinyl-2-pyrrollidinone, N-methacryloylpyrrolidine, and combinationsthereof.
 9. The synthetic co-polymer according to claim 1, wherein saidone or more hydrophilic co-monomer is a selected from the group of:acrylic acid, methacrylic acid, 2-hydroxyethyl methacrylate (HEMA), N,Ndimethylacrylamide, N,N-diethylacrylamide,2-[N,N-dimethylamino]ethylacrylamide,2-[N,N-diethylamino]ethylacrylamide, N,N-diethylmethacrylamide,2-[N,N-dimethylamino]ethylmethacrylamide,2-[N,N-diethylamino]ethylmethacrylamide, 2-vinyl-N-pyrrollidone,2-[N,N-diethylamino]ethylacrylate, 2-[N,N-dimethylamino]ethylacrylate,2-[N,N-diethylamino]ethylmethacrylate,2-[N,N-dimethylamino]ethylmethacrylate, and combinations thereof. 10.The synthetic co-polymer according to claim 1, wherein said one or moreacryl- or methacryl-carboxylic acid co-monomer is selected from thegroup of acrylic acid, methacrylic acid, and substituted versionsthereof, and said cross-linkable moiety is a succinimidyl group, animidazole, a benzotriazole, ap-nitrophenol or2-(N-morpholino)ethanesulphonic acid.
 11. The synthetic co-polymeraccording to claim 2 that comprises N,N-dimethylacrylamide andN-acryloxysuccinimide.
 12. The synthetic co-polymer according to claim 3that comprises N-isopropylacrylamide, acrylic acid andN-acryloxysuccinimide.
 13. A bio-synthetic matrix comprising: (a) thesynthetic co-polymer according to claim 1; (b) a bio-polymer; and (c) anaqueous solvent, wherein said synthetic co-polymer and said bio-polymerare cross-linked through said pendant cross-linkable moiety to form ahydrogel.
 14. The bio-synthetic matrix according to claim 13, whereinthe amount of synthetic co-polymer is between about 0.1% and about 30%by weight, the amount of bio-polymer is between about 0.3% and about 50%by weight and the amount of aqueous solvent is between about 20% andabout 99.6% by weight.
 15. The bio-synthetic matrix according to claim13, wherein said bio-polymer is selected from the group of collagens,denatured collagens, recombinant collagens, gelatin, fibrin-fibrinogen,elastin, glycoprotein, alginate, chitosan, hyaluronic acid, chondroitinsulphate, glycosaminoglycan (proteoglycan), and derivatives thereof. 16.The bio-synthetic matrix according to claim 13 further comprising one ormore bioactive agent.
 17. The bio-synthetic matrix according to claim16, wherein said one or more bioactive agent is covalently bonded tosaid synthetic co-polymer through said pendant cross-linkable moiety.18. The bio-synthetic matrix according to claim 16, wherein saidbioactive agent comprises the pentapeptide having the sequence YIGSR(SEQ ID NO:1).
 19. The bio-synthetic matrix according to claim 16,wherein said one or more bioactive agent is dispersed in said matrix.20. The bio-synthetic matrix according to claim 13, further comprising aplurality of cells dispersed in said matrix.
 21. A method forregenerating tissue in an animal comprising the step of implanting thebio-synthetic matrix according to claim 13 such that said bio-syntheticmatrix acts as a scaffold for tissue regeneration in said animal.
 22. Amethod for replacing damaged or removed tissue in an animal comprisingthe step of implanting the bio-synthetic matrix according to claim 13 insaid animal.
 23. The method according to claim 22, wherein said tissueis skin or part of an organ.
 24. The method according to claim 22,wherein said tissue is a cornea or a part of a cornea.
 25. A method forcoating a surgical implant comprising the step of applying thebio-synthetic matrix according to claim 13 to a surface of said surgicalimplant.
 26. A composition comprising: (a) one or more bioactive agent;(b) the synthetic co-polymer according to claim 1; (c) a bio-polymer;and (d) an aqueous solvent.
 27. A composition comprising: (a) aplurality of cells; (b) the synthetic co-polymer according to claim 1;(c) a bio-polymer; and (d) an aqueous solvent.
 28. The compositionaccording to claim 27, wherein the amount of synthetic polymer isbetween about 0.1% and about 30% by weight, the amount of bio-polymer isbetween about 0.3% and about 50% by weight and the amount of aqueoussolvent is between about 20% and about 99.6% by weight.
 29. Thecomposition according to claim 27, wherein said bio-polymer is selectedfrom the group of collagens, denatured collagens, recombinant collagens,gelatin, fibrin-fibrinogen, elastin, glycoprotein, alginate, chitosan,hyaluronic acid, chondroitin sulphate, glycosaminoglycan (proteoglycan),and derivatives thereof.
 30. The composition according to claim 27,wherein said synthetic co-polymer and said bio-polymer are cross-linked.31. The composition according to claim 27, wherein said bioactive agentis covalently attached to said synthetic co-polymer through said pendantcross-linkable moiety.
 32. The composition according to claim 27, whichis formulated as an injectable solution, wherein said syntheticco-polymer and said bio-polymer are capable of cross-linking to form ahydrogel in vivo.
 33. The composition according to claim 27, which is apre-formed hydrogel.
 34. An implant for use in tissue engineeringcomprising a pre-formed bio-synthetic matrix, said matrix comprising anaqueous solvent and a bio-polymer cross-linked with the syntheticco-polymer according to claim
 1. 35. The implant according to claim 34,wherein said bio-polymer is selected from the group of collagens,denatured collagens, recombinant collagens, gelatin, fibrin-fibrinogen,elastin, glycoprotein, alginate, chitosan, hyaluronic acid, chondroitinsulphate, glycosaminoglycan (proteoglycan), and derivatives thereof. 36.The implant according to claim 34, wherein the amount of syntheticpolymer is between about 0.1% and 30% by weight, the amount ofbio-polymer is between about 0.3% and 50% by weight and the amount ofaqueous solvent is between about 20% and 99.6% by weight.
 37. Theimplant according to claim 34, wherein said bio-synthetic matrixsupports in-growth of nerves.
 38. The implant according to claim 34,further comprising one or more bioactive agent.
 39. The implantaccording to claim 38, wherein said bioactive agent is covalentlyattached to co-polymer through said pendant cross-linkable moiety. 40.The implant according to claim 34, further comprising a plurality ofcells dispersed in said matrix.
 41. The implant according to claim 40,wherein said cells are stem cells or precursor cells.
 42. Use of theimplant according to claim 34 as an artificial cornea.
 43. A process forpreparing a synthetic co-polymer comprising: (a) dispersing one or moreN-alkyl or N,N-dialkyl substituted acrylamide co-monomer, one or morehydrophilic co-monomer and one or more acryl- or methacryl-carboxylicacid co-monomer derivatised to contain a pendant cross-linkable moietyin a solvent in the presence of an initiator; (b) allowing said one ormore N-alkyl or N,N-dialkyl substituted acrylamide co-monomer, one ormore hydrophilic co-monomer and one or more acryl- ormethacryl-carboxylic acid co-monomer to polymerise to form a syntheticco-polymer, and (c) optionally purifying said synthetic co-polymer. 44.A process for preparing a bio-synthetic matrix comprising the steps of:(a) preparing a synthetic co-polymer by the process according to claim43; (b) dispersing said synthetic co-polymer and a bio-polymer in anaqueous medium; and (c) allowing said synthetic co-polymer and saidbio-polymer to cross-link to provide said bio-synthetic matrix.
 45. Theprocess according to claim 43, wherein the N-alkyl or N,N-dialkylsubstituted acrylamide co-monomer and the hydrophilic co-monomer are thesame.
 46. The process according to claim 43, wherein the N-alkyl orN,N-dialkyl substituted acrylamide co-monomer and the hydrophilicco-monomer are different.
 47. The process according to claim 44, furthercomprising mixing said synthetic co-polymer with one or more bioactiveagent prior to step (b) and allowing said bioactive agent to cross-linkto said synthetic co-polymer through said pendant cross-linkable moiety.48. The process according to claim 44, further comprising mixing saidsynthetic co-polymer and said bio-polymer with a plurality of cells instep (b).
 49. A synthetic co-polymer produced by the process accordingto claim
 43. 50. A bio-synthetic matrix produced by the processaccording to claim
 44. 51-110. (canceled)
 111. The synthetic co-polymeraccording to claim 1, wherein said alkyl or lower alkyl is cycloalkylgroup having between three and six carbon atoms.
 112. The compositionaccording to claim 26, wherein the amount of synthetic polymer isbetween about 0.1% and about 30% by weight, the amount of bio-polymer isbetween about 0.3% and about 50% by weight and the amount of aqueoussolvent is between about 20% and about 99.6% by weight.
 113. Thecomposition according to claim 26, wherein said bio-polymer is selectedfrom the group of collagens, denatured collagens, recombinant collagens,gelatin, fibrin-fibrinogen, elastin, glycoprotein, alginate, chitosan,hyaluronic acid, chondroitin sulphate, glycosaminoglycan (proteoglycan),and derivatives thereof.
 114. The composition according to claim 26,wherein said synthetic co-polymer and said bio-polymer are cross-linked.115. The composition according to claim 26, wherein said bioactive agentis covalently attached to said synthetic co-polymer through said pendantcross-linkable moiety.
 116. The composition according to claim 26, whichis formulated as an injectable solution, wherein said syntheticco-polymer and said bio-polymer are capable of cross-linking to form ahydrogel in vivo.
 117. The composition according to claim 26, which is apre-formed hydrogel.